由托马斯·F.和凯特·米勒·杰弗里斯MEMORIA信托（以DEC）和美国国立卫生研究院授予R35GM119617（以DEC）这项工作是支持的。共焦显微镜成像在所述纳米材料VCU表征核心（NCC）设施执行。

1.获取Nesprin-2G传感器DNA和其它质粒DNA <ol><li>从商业来源获得Nesprin-2G TS（张力传感器），Nesprin-2G HL（无头）控制，mTFP1，金星和TSmod。传播的所有DNA的质粒，并使用标准的大肠杆菌菌株，如DH5-α，净化他们如先前12,13说明。 </li></ol> 2.转染细胞与Nesprin-2G和其它质粒DNA <ol><li>生长NIH 3T3成纤维细胞的细胞至70-90％铺满在6孔细胞培养皿中在标准的细胞培养培养箱中温（37℃）和CO 2（5％）调节。对于细胞生长培养基，用Dulbecco氏补充有10％胎牛血清的改良的Eagle培养基（DMEM）。 </li><li>在细胞培养罩，除去培养基并用约1mL降低血清细胞培养基冲洗每一个孔。添加降低血清细胞培养基中的800μL到每个孔并放置在培养箱中在6孔腔。 </li><li>吸移管700降低血清细胞培养基到1.5mL管中的脂质载体溶液35μL的μL以形成"lipomix"。通过移液混合。标记管具有"L"。 </li><li>收集6个的1.5mL管中并标记它们1到6吸取100μL的降低血清细胞培养基到每个管中。 <ol><li>从步骤1中，移液管2 Nesprin 2G-TS的微克使用质粒DNA的浓度成管1和2。移液器2 Nesprin-HL的微克到管中3和4移取1 MTFP的微克到管5移液器1微克mVenus的入管6吹打不同类型的DNA时，不要再使用移液管尖端。 </li></ol></li><li>移液管将100μL从"L"管到每个标记的管（1-6）的lipomix的，并通过反复吹打混匀。使用干净的吸管每个管。孵育10-20分钟。 </li><li>从每个标记的管加入200微升到井在6孔室具有70-90％汇合的细胞。标签的每个井的顶部与相应的DNA溶液。放置6孔室在4-6 h的培养箱中。 </li><li>吸出介质，并添加减少的血清细胞培养基中的1-2毫升冲洗。吸出降低血清细胞培养基，加入胰蛋白酶的2 mL至每个孔中，并将其放置在6孔培养皿在培养箱（5-15分钟）。 </li><li>虽然细胞在培养箱中分离，涂层6玻璃底在溶解于磷酸盐缓冲盐水（PBS）20微克/毫升的浓度与纤连蛋白的一个层观看菜肴。允许的菜涂覆在细胞培养罩（约20分钟）的表面上。 </li><li>加入2毫升的DMEM一旦细胞被分离中和胰蛋白酶。 </li><li>传送每个孔中的物质与标记的15-mL离心管中，并在摇摆转子离心机降速在90×g离心5分钟。吸出上清液并重新悬浮用移液管混合在DMEM 1000微升的各细胞沉淀。 </li><li>抽吸从纤连蛋白混合物玻璃菜肴和吸管每个细胞悬浮液的1000μL到玻璃培养皿。 </li><li>后的细胞沉淀到的玻璃盘的底部（〜15分钟），在细胞培养箱中添加另外1毫升的DMEM + 10％FBS + 1％青霉素 - 链霉素向每个孔和地点。允许细胞附着并表达传感器，用于至少18-24小时。 注:电池使用商用阳离子脂质转染试剂（见材料的表 ）转。可替代地，稳定的细胞系可以通过使用质粒与基因赋予对毒素进行选择（所述的pcDNA质粒为Nesprin-TS和-HL上可用的DNA库网站（参见材料的表 ）提供表达遗传霉素抗性细胞）。此外，病毒感染的方法（慢病毒，逆转录病毒，或腺病毒）可用于表达在，是很难转染细胞的传感器。 </li><li>除了Nesprin-TS，转染的细胞附加与Nesprin HL零为CE控制，如在以下步骤中描述2.1-2.12; TSmod也可以用作零力控制并且应当表现出相似的FRET到Nesprin-HL。 </li><li>转染细胞用mTFP1和金星，以产生频谱指纹（参见步骤4）。 注:mTFP1和金星通常表达水平高于Nesprin-TS，并且同样地，低量的DNA可能需要被转染的，以实现相似的表达水平。 </li></ol> 3.确认转染效率<ol><li>大致完成转染后18-24小时，使用倒置荧光显微镜通过荧光细胞的数目相比较，以在视图细胞（通常为5-30％）的总数来检查转染的效率。 <ol><li>使用配备有接近462纳米（mTFP1）或525纳米（金星）和近492纳米（mTFP1）或525纳米（金星）为中心的发射滤波器的激励频率的荧光显微镜。 注:另外，GFP过滤器组将捕获mTFP1的组合，并且已经NUS排放和允许的转染效率的确认。 </li></ol></li><li>转染后48个小时内的图像的活细胞。 <ol><li>可替代地，在固定的4％多聚甲醛（与钙和镁的PBS中）5分钟，储存于PBS中，视图细胞48小时后8。 注意:超过48小时，信号质量和强度衰减。固定细胞保持它们的状态，其中包括FRET中表达8;然而，细胞只应在PBS成像，如固定介质可能影响FRET 14。固定的细胞仅可相对于其他固定的细胞，因为有可能在表达的变化。 注意:多聚甲醛是有毒的。穿戴合适的个人防护装备（PPE）。 </li></ol></li></ol> mTFP1和金星荧光基团的4捕捉光谱指纹光谱分离<ol><li>在细胞培养罩，更换成像mediu细胞培养基米（HEPES缓冲的），补充有10％胎牛血清。 </li><li>放置观看碗碟的温度受控（37℃）共聚焦显微镜阶段。 </li><li>将带有mTFP1转染的细胞在油物镜的玻璃的观看菜在60X放大倍数为1.4的数值孔径。 注:此油经激光扫描显微镜中使用的60X物镜到接近于玻璃基板的折射率。油放置在物镜的顶部并且与玻璃盖玻片直接接触。 </li><li>定位mTFP1表达细胞与458 nm激发源，并在500纳米为中心的发射带通滤波器。 </li><li>随着视场荧光细胞，选择（在这里所使用的软件"LAMBDA模式"）的光谱检测模式和捕获分光图像（ 图3-1）;包括除了使用10度纳米的增量（ 图3-3）458nm处的所有频率。选择明亮fluoresc在电池（20像素的半径）ENT区域（ROI）。 注意:mTFP1表达ROI的频谱形状应保持在单元相对恒定。如果该形状变化很大，重新调整激光和增益的设置，以改善信噪比。 </li><li>通过单击添加荧光ROI的意思是，归一化强度的光谱数据库"光谱保存到数据库中。" </li><li>优化激光功率和增益，使得良好的信噪比得以实现。设定适用于不同的设备而有所不同。使用非荧光，未转染细胞作为背景参考，提高增益和功率，直到所述细胞是明亮的，但不超过检测器（饱和点）的动态范围。背景细胞不应该在平均后检测的荧光。 </li><li>重复此过程，金星转染的细胞，但下列情况除外: <ol><li>确保激励频率是在515纳米和该带通滤波器是CE定位金星表达细胞时接近530纳米ntered。 </li><li>在"光谱模式"，使用的515nm的，而不是458纳米的激发频率。 </li></ol></li></ol> 5.捕获未混合的图像<ol><li>切换拍摄模式为"光谱分离。" </li><li>添加金星和mTFP1的光谱指纹到解混信道（ 图3中 ，箭头-2）。 </li><li>设置的激发激光回458纳米氩源。 </li><li>放置60X油浸物镜上面的Nesprin-TS观看菜。 </li><li>聚焦在荧光细胞与足够亮表达后，调整增益和激光功率来优化信噪比。 注意:由于Nesprin-TS的FRET效率接近20％，未混合的金星图像应比未混合mTFP1图像基本上调光器。一旦可接受的功率和增益设置已被迭代地确定，这些参数必须保持所有的图像恒定上校ured。 </li><li>捕捉的最小Nesprin-TS细胞的15-20图像具有相对类似的亮度和避免过度像素饱和（饱和的所有像素的图像处理过程中被除去）。 </li><li>重复使用相同的成像参数Nesprin-HL细胞的图像捕获处理。 </li></ol> 6.图像处理和比率的图像分析<ol><li>使用开源的ImageJ（斐济）软件（http://fiji.sc/），使用BioFormats Reader中打开本机格式的图像。 </li><li>前处理的图像，并计算使用先前建立的方案15的比率的图像。 </li></ol>

<ol>
	<li>Conway, D. E., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-dependent+cellular+mechanotransduction+in+atherosclerosis.">Flow-dependent cellular mechanotransduction in atherosclerosis.</a> J Cell Sci. 126, 5101-5109 (2013).</li><li>Arsenovic, P. T., Ramachandran, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nesprin-2G,+a+Component+of+the+Nuclear+LINC+Complex,+Is+Subject+to+Myosin-Dependent+Tension.">Nesprin-2G, a Component of the Nuclear LINC Complex, Is Subject to Myosin-Dependent Tension.</a> Biophys J. 110 (1), 34-43 (2016).</li><li>Grashoff, C., Hoffman, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+mechanical+tension+across+vinculin+reveals+regulation+of+focal+adhesion+dynamics.">Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics.</a> Nature. 466 (7303), 263-266 (2010).</li><li>Austen, K., Ringer, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+rigidity+sensing+by+talin+isoform-specific+mechanical+linkages.">Extracellular rigidity sensing by talin isoform-specific mechanical linkages.</a> Nat Cell Bio. 17 (12), 1597-1606 (2015).</li><li>Meng, F., Sachs, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+dynamic+cytoplasmic+forces+with+a+compliance-matched+FRET+sensor.">Visualizing dynamic cytoplasmic forces with a compliance-matched FRET sensor.</a> J Cell Sci. 124, 261-269 (2011).</li><li>Borghi, N., Sorokina, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-cadherin+is+under+constitutive+actomyosin-generated+tension+that+is+increased+at+cell-cell+contacts+upon+externally+applied+stretch.">E-cadherin is under constitutive actomyosin-generated tension that is increased at cell-cell contacts upon externally applied stretch.</a> Proc. Natl. Acad. Sci. U.S.A. 109 (31), 12568-12573 (2012).</li><li>Cai, D., Chen, S. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+Feedback+through+E-Cadherin+Promotes+Direction+Sensing+during+Collective+Cell+Migration.">Mechanical Feedback through E-Cadherin Promotes Direction Sensing during Collective Cell Migration.</a> Cell. 157 (5), 1146-1159 (2014).</li><li>Conway, D. E., Breckenridge, M. T., Hinde, E., Gratton, E., Chen, C. S., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+Shear+Stress+on+Endothelial+Cells+Modulates+Mechanical+Tension+across+VE-Cadherin+and+PECAM-1.">Fluid Shear Stress on Endothelial Cells Modulates Mechanical Tension across VE-Cadherin and PECAM-1.</a> Curr Bio CB. 23 (11), 1024-1030 (2013).</li><li>Brenner, M. D., Zhou, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spider+Silk+Peptide+Is+a+Compact,+Linear+Nanospring+Ideal+for+Intracellular+Tension+Sensing.">Spider Silk Peptide Is a Compact, Linear Nanospring Ideal for Intracellular Tension Sensing.</a> Nano Lett. 16 (3), 2096-2102 (2016).</li><li>Rothenberg, K. E., Neibart, S. S., LaCroix, A. S., Hoffman, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+Cell+Geometry+Affects+the+Spatial+Distribution+of+Load+Across+Vinculin.">Controlling Cell Geometry Affects the Spatial Distribution of Load Across Vinculin.</a> Cell Mol Bioeng. 8 (3), 364-382 (2015).</li><li>Ostlund, C., Folker, E. S., Choi, J. C., Gomes, E. R., Gundersen, G. G., Worman, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+and+molecular+interactions+of+linker+of+nucleoskeleton+and+cytoskeleton+(LINC)+complex+proteins.">Dynamics and molecular interactions of linker of nucleoskeleton and cytoskeleton (LINC) complex proteins.</a> J Cell Sci. 122, 4099-4108 (2009).</li><li>Froger, A., Hall, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+plasmid+DNA+into+E.+coli+using+the+heat+shock+method.">Transformation of plasmid DNA into E. coli using the heat shock method.</a> J Vis Exp. (6), e253 (2007).</li><li>Zhang, S., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purifying+plasmid+DNA+from+bacterial+colonies+using+the+QIAGEN+Miniprep+Kit.">Purifying plasmid DNA from bacterial colonies using the QIAGEN Miniprep Kit.</a> J Vis Exp. (6), e247 (2007).</li><li>Rodighiero, S., Bazzini, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation,+mounting+and+sealing+with+nail+polish+of+cell+specimens+lead+to+incorrect+FRET+measurements+using+acceptor+photobleaching.">Fixation, mounting and sealing with nail polish of cell specimens lead to incorrect FRET measurements using acceptor photobleaching.</a> Cell. Physiol. Biochem. 21 (5-6), 489-498 (2008).</li><li>Kardash, E., Bandemer, J., Raz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+protein+activity+in+live+embryos+using+fluorescence+resonance+energy+transfer+biosensors.">Imaging protein activity in live embryos using fluorescence resonance energy transfer biosensors.</a> Nat Protoc. 6 (12), 1835-1846 (2011).</li><li>Vaziri, A., Mofrad, M. R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+and+deformation+of+the+nucleus+in+micropipette+aspiration+experiment.">Mechanics and deformation of the nucleus in micropipette aspiration experiment.</a> J Biomech. 40 (9), 2053-2062 (2007).</li><li>Wang, N., Naruse, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+behavior+in+living+cells+consistent+with+the+tensegrity+model.">Mechanical behavior in living cells consistent with the tensegrity model.</a> Proc. Natl. Acad. Sci. U.S.A. 98 (14), 7765-7770 (2001).</li><li>Nagayama, K., Yahiro, Y., Matsumoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+fibers+stabilize+the+position+of+intranuclear+DNA+through+mechanical+connection+with+the+nucleus+in+vascular+smooth+muscle+cells.">Stress fibers stabilize the position of intranuclear DNA through mechanical connection with the nucleus in vascular smooth muscle cells.</a> FEBS letters. 585 (24), 3992-3997 (2011).</li><li>Luxton, G. W. G., Gomes, E. R., Folker, E. S., Vintinner, E., Gundersen, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+arrays+of+nuclear+envelope+proteins+harness+retrograde+actin+flow+for+nuclear+movement.">Linear arrays of nuclear envelope proteins harness retrograde actin flow for nuclear movement.</a> Science. 329 (5994), 956-959 (2010).</li><li>Chen, Y., Mauldin, J. P., Day, R. N., Periasamy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+spectral+FRET+imaging+microscopy+for+monitoring+nuclear+protein+interactions.">Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions.</a> J Microsc. 228, 139-152 (2007).</li><li>Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nat Protoc. 8 (11), 2281-2308 (2013).</li><li>LaCroix, A. S., Rothenberg, K. E., Berginski, M. E., Urs, A. N., Hoffman, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction,+imaging,+and+analysis+of+FRET-based+tension+sensors+in+living+cells.">Construction, imaging, and analysis of FRET-based tension sensors in living cells.</a> Methods Cell Biol. , (2015).</li></ol>

The authors have nothing to disclose.

一种方法和跨Nesprin-2G，在核LINC复合体的蛋白质的机械张力的活细胞成像演示，在上面概述的。在此之前的工作，各种技术，如微吸管，磁珠术和显微激光烧蚀，已经被用于对细胞核申请应变，并测量它的体积的材料特性16，17，18。 然而，直到我们最近的工作，还没有研究已经直接表明拉力在活细胞中2</...

力敏感，遗传编码的FRET传感器最近出现的用于在活细胞中测量基于拉伸力，提供深入了解的机械力跨蛋白1，2，3，4施加的重要工具。有了这些工具，研究人员能够以非侵入性图像内的力量在使用传统的荧光显微镜的活细胞。这些传感器由通过弹性肽3分离的FRET对（供体和受体荧光蛋白，最频繁蓝色供体和黄色受体）的。在对比C-或N-末端标记，该传感器被插入到测量跨蛋白质传递的机械力的蛋白质的内部位点，表现为一个分子应变计。增加跨越传感器的结果的机械张力在FRET-P之间的距离增加空气，导致降低FRET 3。其结果是，该FRET是负相关的拉伸力。 这些基于荧光的传感器已被用于粘着斑蛋白（纽蛋白3和踝蛋白4），细胞骨架蛋白（α辅肌动蛋白5），和细胞-细胞连接蛋白显影（E-钙粘蛋白6，7，VE-钙粘蛋白8，和PECAM 8）。在这些生物传感器中最常用的和充分表征的弹性连接体被称为TSmod和由40个氨基酸，（GPGGA）8，其从所述蜘蛛丝蛋白衍生鞭的重复序列组成。 TSmod已经显示出表现为线性弹性纳米弹簧，与FRET响应要求1至拉伸力3 5对-N。鞭状的不同长度可以被用于改变所述动态řTSmod FRET力灵敏度9的安格。除了鞭状，血影蛋白重复5和绒毛头戴受话器肽（称为HP35）4已被用作在类似力的生物传感器4 FRET对之间的弹性的肽。最后，最近的一份报告显示，TSmod也可用于检测压缩力10。 我们最近开发了用于细胞核-细胞骨架（LINC）复合蛋白Nesprin2G的接头的力传感器，通过使用TSmod插入称为迷你Nesprin2G（ 图2C），其行为类似于内源性一个先前开发的截短的蛋白Nesprin2G Nesprin-2G 11。在LINC复杂包含了从外面导致细胞核内，连接细胞质细胞骨架的核纤层的多种蛋白质。 Nesprin-2G是结构蛋白结合到两个肌动蛋白骨架在细胞质和核周空间SUN蛋白质。使用我们的生物传感器，我们能够证明Nesprin-2G是受制于NIH3T3成纤维细胞肌动球蛋白2依赖紧张。这是第一次力在核LINC复杂的跨蛋白直接测量，它很可能成为了解力在力学生物学的核心作用的重要工具。 下面的协议提供的如何使用Nesprin-2G力传感器，包括在哺乳动物细胞中Nesprin张力传感器（Nesprin-TS）的表达Nesprin-细胞的FRET图像的表达，以及采集和分析了详细的方法TS。使用配备有光谱检测器的倒共焦显微镜，如何测量敏化发射的描述使用FRET光谱分离，并设置比例FRET成像。输出比率的图像可以被用来制造相对QUAntitative力比较。虽然该协议的重点是Nesprin-TS的成纤维细胞中的表达，这是很容易适应其它哺乳动物细胞，包括细胞系和原代细胞。此外，该协议，因为它涉及到图像采集和分析FRET可以容易地适用于已经被用于其它蛋白开发了其他基于FRET的力生物传感器。

<table border="0" cellpadding="0" cellspacing="0" width="1183">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Nesprin-TS DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">68127</td>
			<td style="width:475px;">Retrieve from https://www.addgene.org/68127/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Nesprin-HL DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">68128</td>
			<td>Retrieve from https://www.addgene.org/68128/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">mTFP1 DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">54613</td>
			<td>Retrieve from https://www.addgene.org/54613/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">mVenus DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">27793</td>
			<td>Retrieve from https://www.addgene.org/27793/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">TSmod DNA</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">26021</td>
			<td>Retrieve from https://www.addgene.org/26021/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Competent Cells</td>
			<td style="width:108px;">Bioline</td>
			<td style="width:119px;">BIO-85026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Liquid LB Media</td>
			<td style="width:108px;">ThermoFisher</td>
			<td align="right" style="width:119px;">10855001</td>
			<td>https://www.thermofisher.com/order/catalog/product/10855001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Solid LB Bacterial Culture Plates</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">L5667</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/l5667?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Ampicillin</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:119px;">A9518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Spectrophotometer</td>
			<td style="width:108px;">Biorad</td>
			<td style="width:119px;">273 BR 07335</td>
			<td>SmartSpec Plus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">quartz cuvette</td>
			<td style="width:108px;">Biorad</td>
			<td align="right" style="width:119px;">1702504</td>
			<td>Cuvette for SmartSpec Plus</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">&#160;DNA isolation kit</td>
			<td style="width:108px;">Macherey-Nagel</td>
			<td align="right" style="width:119px;">740412.5</td>
			<td>NucleoBond Xtra Midi Plus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">6-well cell culture dish</td>
			<td style="width:108px;">Falcon-Corning</td>
			<td align="right" style="width:119px;">353046</td>
			<td>Multiwell 6-well Polystrene Culture Dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Dulbecco&#39;s Modified Eagle Medium, (DMEM) cell media</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">11995-065</td>
			<td>DMEM(1x)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">&#160;Bovine Serum</td>
			<td>Life Technologies</td>
			<td>16170-078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">reduced serum cell media</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">31985-070</td>
			<td>Reduced Serum Medium, &#34;optimem&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Lipid Carrier Solution</td>
			<td style="width:108px;">invitrogen</td>
			<td style="width:119px;">11668-019</td>
			<td>Lipid Reagent, &#34;Lipofectamine 2000&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">1.5mL sterile plastic tube</td>
			<td style="width:108px;">Denville</td>
			<td style="width:119px;">c2170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Trypsin</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td>0.25% Trypsin-EDTA (1x)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">glass-bottom microscope viewing dish</td>
			<td style="width:108px;">In Vitro Scientific</td>
			<td style="width:119px;">D35-20-1.5-N</td>
			<td>35mm Dish with 20mm Bottom Well #1.5 glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Fibronectin</td>
			<td style="width:108px;">ThermoFisher</td>
			<td align="right" style="width:119px;">33016015</td>
			<td>fibronectin human protein,plasma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:108px;">Gibco</td>
			<td style="width:119px;">14190-144</td>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">15mL sterile centrifuge tube</td>
			<td style="width:108px;">Greiner bio-one</td>
			<td align="right" style="width:119px;">188261</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">swinging rotor centrifuge&#160;</td>
			<td style="width:108px;">Thermo electron</td>
			<td style="width:119px;">Centra CL2</td>
			<td>Swinging rotor thermo electron 236</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">cell culture biosafety hood</td>
			<td style="width:108px;">Forma Scientific</td>
			<td align="right" style="width:119px;">1284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">&#160;climate controlled cell culture incubator</td>
			<td style="width:108px;">ThermoFisher</td>
			<td align="right" style="width:119px;">3596</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">inverted LED widefield fluorescent microscope</td>
			<td style="width:108px;">Life technologies</td>
			<td style="width:119px;">EVOS FL</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">Clear HEPES buffered imaging media</td>
			<td style="width:108px;">Molecular Probes</td>
			<td style="width:119px;">A14291DJ</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">Fetal bovine Serum</td>
			<td style="width:108px;">Life technologies</td>
			<td style="width:119px;">10437-028</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:483px;">Temperature Controlled-Inverted confocal w/458 and 515nm laser sources&#160;</td>
			<td style="width:108px;">Zeiss</td>
			<td style="width:119px;">&#160;LSM 710-w/spectral META detector</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:483px;">Outgrowth Media</td>
			<td style="width:108px;">Newengland Biolabs</td>
			<td style="width:119px;">B9020s</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:483px;">NIH 3T3 Fibroblasts</td>
			<td style="width:108px;">ATCC</td>
			<td style="width:119px;">CRL-1658</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol for using f&#246;rster resonance energy transfer (fret)-force biosensors to measure mechanical forces across the nuclear linc complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

按照上述协议，质粒DNA从DNA库中获取并转化入大肠杆菌细胞中。表达该传感器DNA 的大肠杆菌从LB /氨苄青霉素平板上选择并在液体LB肉汤扩增。以下载体的扩增，DNA质粒使用标准，市售的DNA提取试剂盒纯化为TRIS-EDTA缓冲液中。使用分光光度计，纯化的DNA被量化为微克/毫升（ 图1A，1-2天）的标准浓度。 ?...

Watch this Scientific Journal Video about A Protocol for Using FÃƒÂ¶rster Resonance Energy Transfer FRET-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex at JoVE.com

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

一种利用共振能量转移（FRET）的生物传感器-force议定书衡量所有核LINC复杂的机械力

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

a-protocol-for-using-frster-resonance-energy-transfer-fret-force

Research

JoVE Journal

Bioengineering

10.5K 次观看.  Virginia Commonwealth University. A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex. 

视频: A Protocol for Using Förster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

科研

生物工程

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique where X-rays are elastically scattered by an inhomogeneous sample in the nm-range at small angles (typically 0.1 - 5&deg;) and wide angles (typically &gt; 5&deg;). This technique provides information about the shape, size, and distribution of macromolecules, characteristic distances of partially ordered materials, pore sizes, and surface-to-volume ratio. Small Angle X-ray Scattering (SAXS) is capable of delivering structural information of macromolecules between 1 and 200 nm, whereas Wide Angle X-ray Scattering (WAXS) can resolve even smaller Bragg spacing of samples between 0.33 nm and 0.49 nm based on the specific system setup and detector. The spacing is determined from Bragg's law and is dependent on the wavelength and incident angle.
In a SWAXS experiment, the materials can be solid or liquid and may contain solid, liquid or gaseous domains (so-called particles) of the same or another material in any combination. SWAXS applications are very broad and include colloids of all types: metals, composites, cement, oil, polymers, plastics, proteins, foods, and pharmaceuticals. For solid samples, the thickness is limited to approximately 5 mm.
Usage of a lab-based SWAXS instrument is detailed in this paper. With the available software (e.g., GNOM-ATSAS 2.3 package by D. Svergun EMBL-Hamburg and EasySWAXS software) for the SWAXS system, an experiment can be conducted to determine certain parameters of interest for the given sample. One example of a biological macromolecule experiment is the analysis of 2 wt% lysozyme in a water-based aqueous buffer which can be chosen and prepared through numerous methods. The preparation of the sample follows the guidelines below in the Preparation of the Sample section. Through SWAXS experimentation, important structural parameters of lysozyme, e.g. the radius of gyration, can be analyzed.

The demonstration of the small and wide angle X-ray scattering (SWAXS) procedure has become instrumental in the study of biological macromolecules. Through the use of the instrumentation and procedures of specific angle methods and preparation, the experimental data from the SWAXS displays the atomic and nano-scale characterization of macromolecules.

小型和广角X-射线散射研究生物大分子的解决方案

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique ...

Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein activity and have diverse roles in many biological processes. For example, SUMO covalently modifies a large number or proteins with important roles in many cellular processes, including cell-cycle regulation, cell survival and death, DNA damage response, and stress response 1-5. SENP, as SUMO-specific protease, functions as an endopeptidase in the maturation of SUMO precursors or as an isopeptidase to remove SUMO from its target proteins and refresh the SUMOylation cycle 1,3,6,7.
The catalytic efficiency or specificity of an enzyme is best characterized by the ratio of the kinetic constants, kcat/KM. In several studies, the kinetic parameters of SUMO-SENP pairs have been determined by various methods, including polyacrylamide gel-based western-blot, radioactive-labeled substrate, fluorescent compound or protein labeled substrate 8-13. However, the polyacrylamide-gel-based techniques, which used the "native" proteins but are laborious and technically demanding, that do not readily lend themselves to detailed quantitative analysis. The obtained kcat/KM from studies using tetrapeptides or proteins with an ACC (7-amino-4-carbamoylmetylcoumarin) or AMC (7-amino-4-methylcoumarin) fluorophore were either up to two orders of magnitude lower than the natural substrates or cannot clearly differentiate the iso- and endopeptidase activities of SENPs.
Recently, FRET-based protease assays were used to study the deubiquitinating enzymes (DUBs) or SENPs with the FRET pair of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) 9,10,14,15. The ratio of acceptor emission to donor emission was used as the quantitative parameter for FRET signal monitor for protease activity determination. However, this method ignored signal cross-contaminations at the acceptor and donor emission wavelengths by acceptor and donor self-fluorescence and thus was not accurate.
We developed a novel highly sensitive and quantitative FRET-based protease assay for determining the kinetic parameters of pre-SUMO1 maturation by SENP1. An engineered FRET pair CyPet and YPet with significantly improved FRET efficiency and fluorescence quantum yield, were used to generate the CyPet-(pre-SUMO1)-YPet substrate 16. We differentiated and quantified absolute fluorescence signals contributed by the donor and acceptor and FRET at the acceptor and emission wavelengths, respectively. The value of kcat/KM was obtained as (3.2 &plusmn; 0.55) x107 M-1s-1 of SENP1 toward pre-SUMO1, which is in agreement with general enzymatic kinetic parameters. Therefore, this methodology is valid and can be used as a general approach to characterize other proteases as well.

Quantitative FRET F&#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

A novel method involving quantitative analysis of FRET (F&ouml;rster Resonance Energy Transfer) signals is described for studying enzyme kinetics. KM and kcat were obtained for the hydrolysis of the catalytic domain of SENP1 (SUMO/Sentrin specific protease 1) to pre-SUMO1 (Small Ubiquitin-like MOdifier). The general principles of this quantitative-FRET-based protease kinetic study can be applied to other proteases.

定量荧光共振能量转移（福斯特共振能量转移）SENP1蛋白酶的动力学测定分析

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

组织工程生物反应器的双轴机械装载设计

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

使用原子力显微镜测量活细胞的力学性能

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a F&ouml;rster resonance energy transfer (FRET) probe in which the &epsilon; subunit of the FoF1-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the &epsilon; subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.

We describe the use of two ratiometric, genetically encoded biosensors, which are based on GFP, to monitor mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells.

成比例的生物传感器测量活酵母细胞中的线粒体氧化还原状态和ATP

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell ...

From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data

Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g., signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation.
The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data, we propose a triage scheme that categorizes both objective data set characteristics and subjective personal criteria for the analysis of the different data sets.

The bottleneck for cellular 3D electron microscopy is feature extraction (segmentation) in highly complex 3D density maps. We have developed a set of criteria, which provides guidance regarding which segmentation approach (manual, semi-automated, or automated) is best suited for different data types, thus providing a starting point for effective segmentation.

从体素知识:实用指南复电镜三维数据的分割

Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

协议生物膜形成的流光在微流体器件与微柱

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

荧光共振能量转移研究构象变化的膜蛋白在哺乳动物细胞中表达

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (&#956;CP) is a versatile technique to pattern such grafted PEG brushes, but manual &#956;CP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-&#956;CP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal &#8216;click&#8217; chemistries. Here, we describe in detail the workflow to manufacture such substrates.

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &#181;CP and Sequential Nucleophilic Substitution

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

制造复杂的栽培基质使用机器人微接触印刷（R-μCP）和顺序亲核取代

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...