本教程介绍了在国家犹太健康进行和发展的质谱核心设施内。该NJH MS基金是由CCSTI UL1 TR000154支持的一部分。来自美国国立卫生研究院拨款资助P20 HL-113445和R01 HL-095432也支持这项工作。

1，初步考虑，文书和标准的制备<ol><li>总是用玻璃用于存储（玻璃培养管，玻璃自动进样瓶）或转移（玻璃吸管）脂类和有机溶剂。 </li><li>最小化所有的脂质样品和标准暴露在空气中。密封聚四氟乙烯（PFTE）毛帽紧密，以避免暴露于空气和蒸发。立刻干重悬血脂在未来的溶剂或让他们在稳定的氮气流。 </li><li>用100微升的样品。倘或更多（或更少）的样品是可用的，相应地调整音量。然而在NH 2 SPE柱脂分馏时，表示卷必须保持所示。 </li><li>打开离心机，并设置为0°C至开始样品制备前。 </li><li>这种技术使用了许多挥发性溶剂所以保持样品制备过程中皑皑所有溶剂。 </li><li>准备两种类型的每个建议标准。 </li><li>准备一个阴性对照包括尖刺，在所有的标准在恒定的浓度。这是掺入的所有样品以及一个集中的样品被用作一个批次QC（用于监测在不同天的样品制备重现性掺入合并的样品）和仪器QC（尖刺汇集的样品预先制备，分等分，并用于在分析过程中，并在不同的日子监测仪器条件/波动）。 <ol><li>在1倍，2倍和4倍标准尖峰准备阳性对照。阳性对照加入到所有样品以及一个收集的血浆样品，并作为质量控制的化合物为样品制备步骤和仪器分析步骤，分析和确定在数据分析期间的倍数变化的差异，以确保在所有方面准备和仪器分析被正确执行。 </li></ol></li><li>店标在-20°C和样品储存在-80°C。某些内部立场ARDS以下长期贮存，便很容易降解应贮存于-80℃。 </li><li>此过程需要使用有害，易燃或挥发性溶剂，如甲基叔丁基醚。执行在通风橱中的所有步骤。 </li></ol> 2，内部标准<ol><li>请根据各个项目及其具体的实验设计内部标准（ISTD）。对于生物体液如血浆，支气管肺泡灌洗液，或尿，同位素标记的内标在性质上与这些样品中的生物发现了类似的将是理想的。这些将包括但不限于氨基酸，激素或脂质。对植物代谢的样品，如类黄酮，类胡萝卜素或标记的标准可以被使用。这同样适用于其它代谢研究由此研究者应该选择的内标是代表性的样品类型进行了分析。 </li><li>确保所选择的内标涉及面广的色谱;例如，如果t他的采集时间为20分钟，即洗脱，每5分钟的标准可以被使用。 </li><li>在2毫克/毫升使用各种同位素标记的标准和/或其它极性化合物，它们是外源到被分析的样品建立亲水储备溶液。从各原液，建立在1:1的甲醇溶液1：水与所有标准，以25μg/ ml的肌酸酐-D 3的最终浓度100微克/毫升赖氨酸-D 4，和200微克/毫升缬氨酸-D 8 。 </li><li>创建使用各种同位素标记的标准和/或其它非极性化合物，其是外源到被分析的样品的疏水性储备液;库存浓度的17时00脂肪酸（4毫克/毫升），19:1脂肪酸（4毫克/毫升），17点00分的神经酰胺（2毫克/毫升），17点00的PE（1.75毫克/毫升），15 ：0 PC（2毫克/毫升）和睾酮-D 2（1毫克/毫升）。从每个股票，建立在1:1的氯仿一个解决方案：甲醇与所有标准的50的终浓度μg/ ml的17时00分的神经酰胺，100μg/ ml的15时的PC，100μg/ ml的睾酮-D 2，200微克/毫升17点00脂肪酸，200μg/ ml的19点01脂肪酸，和200微克/毫升17:0的PE在标准混合。 </li><li>使用至少五个不同浓度的各项准则，以确定检测仪器对这些化合物的线性度的极限测试每个标准的电离程度提前。原液为每个单独的标准溶液的浓度将取决于实验设计而有所不同，并且在组合的加标标准各标准溶液的浓度范围可为20微克/毫升至2毫克/毫升，这取决于它们怎样以及电离。 </li><li>创建阳性对照的秒杀组合，并将它们添加到1X，2X，4X或浓度水平的样品，监测定量的样品制备和仪器数据的准确性的实力。阳性对照的终浓度可是：2毫克/毫升的D-葡萄糖，100微克/毫升丙氨酸-D 3，200μg/ ml的甲基丙二酸-D 3，20μg/ ml的甘油三酯-D 5，和/或其它疏水性和亲水性的标准。调整的基础上用于分析的MS和HPLC仪器的灵敏度的标准浓度。 </li></ol> 3，蛋白质沉淀<ol><li>解冻样品至室温，穗10微升内标法对每个样品，如下所述。 </li><li>秒杀10微升亲水和疏水标准溶液（从步骤2.3和2.4的股票创建），以每个样品。根据需要调整以用于分析的MS和HPLC仪器的灵敏度的标准浓度<ol><li>秒杀10微升无论是1X，2X，4X或阳性对照溶液（从步骤2.6股票创建）到每个样品。根据需要调整基于MS和HP的敏感性的标准浓度用于分析的LC仪器</li><li>涡流每个样品10秒前的蛋白沉淀步骤</li></ol></li><li>添加400μl的冰冷的甲醇中（储存于-20℃）到各样品中。 </li><li>涡旋每管10秒。 </li><li>离心机在18,000×g的0°C下15分钟。 </li><li>传输所有上清液至新的玻璃培养管中，然后在N 2下干燥。 </li><li>如果分析的蛋白质沉淀中，请继续步骤3.8-3.11。如果没有分析的蛋白沉淀，跳到第4节。 注：蛋白质沉淀可含有具有高疏水性化合物进行药物研究时，这将是有益的18，食品分析，涉及相关的疾病，其中非常疏水性化合物的积累，如神经元蜡样-lipofuscinoses 20和溶酶体脂质贮积疏水黄酮19或代谢组学研究疾病21。 </li><li>加入1 ml的MTBE到t他的白色（或灰白色）蛋白沉淀，涡旋每管30秒，然后在18,000离心机在0°C下15分钟×g的。滗出的MTBE层到一个新的玻璃培养管中。 这是因为这里的错误会极大地影响结果的关键步骤（参见图5中有代表性的结果）。 <ol><li>始终如一地吸MTBE相同量对于所有的样品，因为颗粒的大小将样品而异。因此，如果仅900微升可以对样品进行倾析，用最少的上清液，然后滗900微升用于所有样品。 </li></ol></li><li>重复步骤3.9，将有机层合并，以在步骤3.7中制备的相同的玻璃培养管中。 </li><li>干燥的样品由N 2流，重悬在200μl1:1的氯仿：甲醇。轻轻振荡。 </li><li>转移到一个离心管中。离心机在0°C下15分钟在18,000×g离心，然后转移上清液用玻璃自动进样器螺旋盖瓶移液器。 </li></ol> 4，液 - 液萃取<ol><li>使用玻璃移液管，加3毫升甲基叔丁基醚，以干燥后的残留甲醇（从第3，第6步），涡旋30秒，加入750微升的水，然后涡旋每管10秒。 </li><li>旋转〜200×g离心10分钟，离心分离机在RT。 </li><li>吸把2.5ml甲基叔丁基醚层（没有得到水），并转移到一个干净的玻璃培养管。 注：这是因为这里的差异会影响结果的关键一步。 2.5 MTBE中的溶液应该从顶层小心倾出，因为它允许在不吸取水层下面将滗出固定体积。如果样品在实验开始时的体积小于所指示的100微升此方法中，缩放MTBE的体积成比例地反映这一起始样品体积。 </li><li>加3毫升甲基叔丁基醚对样品的剩余的水部分，每管涡旋10秒。 </li><li>旋〜200xg下在离心机在室温下10分钟。 </li><li>吸3毫升甲基叔丁基醚（没有得到水），并结合MTBE管。 </li><li>通过在N 2下干燥，浓缩剩余的水层。 </li><li>在100微升的水重悬的残留物。 </li><li>加入400μl冰冷的甲醇玻璃培养管，涡片刻，然后转移到离心管中。 </li><li>留在-80℃下20-30分钟。自旋为0℃，15分钟18,000×g的。 注：建议把整个样品架子在-80℃下冷冻，因为这将允许任何残留的蛋白沉淀出的甲醇。如果一个-80°C冰箱不可用，其他的选项有：存放于-20℃，将样品在干冰，或使它们在一个冰桶。它一致地存储该样品在寒冷的环境中，并在相同温度下，以允许沉淀是重要的。 </li><li>吸450微升上清，转移到一个干净的离心管中。在真空离心浓缩干燥完全在不超过45°C。 （需时约1-2小时）。 </li><li>重悬干燥上清液中加入200μl5％乙腈/水。轻轻振荡。冷冻在-80°C。 </li></ol> 5，固相萃取<ol><li>干燥氮气氛下将MTBE级分在35℃下用氮气的良好的流动性（大约需要10-15分钟）。 </li><li>当完全干燥后，停止氮气流，并迅速用玻璃移液管在1ml氯仿（ 三氯甲烷 ）重悬。轻轻振荡。 注：溶剂如三氯甲烷具有低的粘度。在移液管，低表面张力使从吸管溶剂损失。建议枪头被预先润湿至少两次，以允许被吸出溶剂，并在移液管的空间之间的平衡。甲气密注射器也可以（如果可用）使用。 </li><li>成立SPE真空歧管和NH 2 </子&gt;分馏固相萃取柱。 </li><li>温暖的样品至室温，并始终不断的氮气源源不断暂停。升温至室温，将允许类脂再悬浮和氮将防止氧化和脂质的聚合。 </li><li>洗净条件SPE柱2个400μL正己烷。丢弃的废物，然后换上新的玻璃收集管。 </li><li>加入样品固相萃取柱，收集流过玻璃管。 </li><li>用玻璃吸管加入1毫升2:1 氯仿 ：异丙醇，收集流过相同的玻璃试管（这是中性分数）。 </li><li>干在N 2中性部分，以减少氧化（大约需要10-15分钟）。 </li><li>用玻璃移液管加入1毫升5％乙酸在乙醚中，收集流过的新的玻璃试管（这是脂肪酸组分）。 </li><li>干在N 2中的脂肪酸比例，以减少氧化（大约需要10-15分钟）。 </li><li>使用塑料提示添加800＆＃181;升的甲醇，以固相萃取柱，并收集流过的15ml塑料锥形管（这是磷脂质）。 </li><li>转移磷脂组分到1.5 ml离心管中。干燥用真空离心浓缩器将样品在45℃（约需1-1.5小时）。 </li><li>重悬分别来自三个组分在200微升100％甲醇，旋涡和转让的样本，以自动进样瓶旋盖储存。 </li></ol> 6，样品储存条件<ol><li>存储所有的样品在-80°C，直到准备好仪器分析。 注意：提取样品，重新溶解在有机溶剂中可被存储于-20℃。然而，这是不建议作为潜在的利益代谢产物会降低在此温度下。液氮储存也是不建议作为研究者已经报道的污染问题，需要特殊储存瓶，并且在腔CAU缺乏一致性唱的很宽的温度波动。 </li><li>如果样品量小（&lt;100微升），请使用小瓶插入，以防止溶剂挥发顶部空间储存过程中。 </li><li>避免冻融样品。解冻的样品只有一次，仪器分析之前。反复冷冻解冻导致样品降解。 </li></ol> 7，液相色谱条件<ol><li>使用C-17 2.1毫米×50毫米（1.8微米）分析柱与C-182.1毫米×12.5毫米（5微米）保护柱来分析疏水性部分。 </li><li>设置自动进样器温度为4 &#160; ℃，柱温：60 &#160; ℃，进样量为2微升，流速0.25毫升/分钟。 </li><li>用0.1％甲酸的水为流动相A和0.1％甲酸的异丙醇：乙腈：水（60:36:4）为流动相B。 </li><li>运行下面的梯度洗脱曲线：开始吨，30％B，并增加至70％B，从0到1分钟，然后从1增加到100％B至15分钟，并保持5分钟，然后用5分钟，10％B洗涤5分钟后运行。 </li><li>使用HILIC2.1毫米×50毫米（2.6微米）分析柱与保护柱来分析亲水组分。 </li><li>设置自动进样器温度为4 &#160; ℃，柱温：20 &#160; ℃，进样量为2微升，流速0.5毫升/分钟。 </li><li>用50％乙腈的10mM乙酸铵，pH值5.8流动相A，并在10 mM醋酸铵pH值5.8为移动相B = 90％乙腈</li><li>运行以下梯度洗脱曲线：开始于从0到2分钟100％B，然后降低到50％B，2至15分钟，然后用5分钟0％B洗涤10分钟后运行。 </li></ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+sample+preparation+approaches+for+phospholipids+profiling+in+human+serum+by+liquid+chromatography&#8211;tandem+mass+spectrometry.">Comparison of sample preparation approaches for phospholipids profiling in human serum by liquid chromatography&#8211;tandem mass spectrometry.</a> Journal of Chromatography A. 1240, 21-28 (2012).</li><li>Michopoulos, F., Lai, L., Gika, H., Theodoridis, G., Wilson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UPLC-MS-Based+Analysis+of+Human+Plasma+for+Metabonomics+Using+Solvent+Precipitation+or+Solid+Phase+Extraction.">UPLC-MS-Based Analysis of Human Plasma for Metabonomics Using Solvent Precipitation or Solid Phase Extraction.</a> Journal of Proteome Research. 8, 2114-2121 (2009).</li><li>Kerns, E. H., Di, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drug-like Properties: Concepts, Structure Design and Methods: from ADME to toxicity optimization. First edn. , (2008).</li><li>Manach, C., Scalbert, A., Morand, C., R&#233;m&#233;sy, C., Jim&#233;nez, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyphenols:+food+sources+and+bioavailability.">Polyphenols: food sources and bioavailability.</a> American Journal of Clinical Nutrition. 79, 727-747 (2004).</li><li>Weimer, J. M., Kriscenski-Perry, E., Elshatory, Y., Pearce, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuronal+ceroid+lipofuscinoses.+Mutations+in+different+proteins+result+in+similar+disease.">The neuronal ceroid lipofuscinoses. Mutations in different proteins result in similar disease.</a> NeuroMolecular Medicine. 1, 111-124 (2002).</li><li>Schulze, H., Sandhoff, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomal+lipid+storage+diseases.">Lysosomal lipid storage diseases.</a> Cold Spring Harbor Perspectives in Biology. 3, (2011).</li><li>Kuhn, E., 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=Interlaboratory+evaluation+of+automated,+multiplexed+peptide+immunoaffinity+enrichment+coupled+to+multiple+reaction+monitoring+mass+spectrometry+for+quantifying+proteins+in+plasma.">Interlaboratory evaluation of automated, multiplexed peptide immunoaffinity enrichment coupled to multiple reaction monitoring mass spectrometry for quantifying proteins in plasma.</a> Molecular and Cellular Proteomics. 11, (2012).</li><li>Rist, M. 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作者宣称，他们有没有竞争的财务权益。

临床代谢组学研究中的一个目标是确定的变化相关的疾病或治疗的代谢。因此，样品制备技术必须是健壮的，一致的和可转让从技术员到技术员，从实验室到实验室22。由此产生的数据需要具有代表性的样品，并确定了需要改变以反映样本集，而不是样品制备误差。因此准确的移液，正确的温度，可混溶层的高效调迁，在氮气下干燥，并用玻璃器皿和技巧同样品牌和大小都是必要的。 在蛋白质沉淀步骤中，至关重要的是，的溶液相同的量从每丸倒出。这减少的变化量，因此降低了变异的样本数据。这种蛋白质沉淀步骤是必需的代谢组学研究中，不能被跳过，因为它消除了蛋白质从之前，小分子的样品谱分析的质谱仪。它消除病原体和大分子，并释放蛋白7必将代谢产物。缺乏蛋白质积累在样品中的扩展了的HPLC柱的寿命并提高了精确度和结果的质量。 图5是对血浆样品进行该方法时所形成的蛋白质沉淀物的描述。这允许小分子的检测，提高了离子丰度，并减少了从样品中蛋白质的基质效应。此外，由于假定所有的蛋白质在该步骤中被除去，从而在LC-MS分析检测到的氨基酸将来源于代谢的变化，而不是从蛋白质的分解。 由于分隔的亲水性和疏水性的代谢产物为两个不相混溶的层的液-液萃取步骤是关键的， 图6示出了LLE程序和一个代表resentation的LLE层。不当的分离两层导致代谢产物要么被丢失或者被洗脱在这两个部分。这一步小心应用降低其显示在疏水部分的亲水性化合物的数量。结果这些化合物变得不可靠，因为它不能确定哪部分含有的代表性结果。当正确完成，代谢产物的重叠降低。 为了防止氧化降解，尤其是在脂类而且在小分子中可以含有的巯基，例如，暴露于氧气，必须保持在最低限度。因此，在氮气始终执行此程序，以减少/防止脂质或含巯基化合物的氧化。此外，样品和/或解决方案的传输速度快（在第一分钟），以减少氧气接触，然后样品在氮气源源不断的迅速放入干倒。一旦干燥后，它们是immediately重新悬浮于100％甲醇中的以上所讨论的原因。 实验室可以受益于许多方面这种全面的方法;研究人员希望找出一个类化合物可以选择最适合自己需要的方法的一部分。那些寻求只执行一种蛋白质沉淀获得的代谢物池可以这样做。如果亲水性代谢物是理想的，例如许多药品，氨基酸和糖，或者，如果仅疏水性代谢物是理想的，如甘油三酯，环氧化物，脂溶性维生素和磷脂，例如，则研究人员可进行液 - 液萃取步骤如下蛋白质沉淀并丢弃不想要的部分。谁需要进一步的子分类的疏水性化合物（中性脂质，脂肪酸和磷脂）的研究人员可以进入到分馏步骤​​。 存储的考虑是重要的maintaini纳克的样品的可行性供以后分析。如果样品保存不当，降解或分解可能发生。理想情况下，样品应避光储存在螺丝帽琥珀色小瓶，防止光敏感品种退化。样品也应保持冷冻于-80℃，以防止代谢降解23-25。虽然在这里详细不讨论的，样品总是在LC-MS分析保存在4℃的自动进样器托盘。这确保了所有的样品都保持在恒定的温度和环境温度变化不影响粘度，溶解性，或样品的稳定性。建议在此过程中的人工环节，如LLE和SPE，为了获得自信和舒适性所涉及的步骤来实施。 一些限制这种技术存在的。疏水性和亲水性代谢物的分离谨慎不保证某些化合物将固有ently分隔成两部分，由于其化学成分和充电状态。此外如在图4中 ，不正确的技术在蛋白质沉淀提取步骤可导致两个样品和质量控制不佳的代谢物可重复性所示。这会影响到统计数据，特别是在小数据集，因为统计力量不可用。因此，至关重要的是，此步骤可以确切地对每个样品相同的每次执行的。另一个限制是时间。虽然有止损点在本协议，其中样品可以冻结，准备接下的一天，一整天，应预留执行此过程。第三，没有一个生物样品中的每一个化合物可以评价为离子抑制。既然是无法确定如何矩阵是影响每个人的代谢产物，目前的选择是评估内部标准，理论上模仿一些类endogenou的s代谢产物。最后，绝对标识不能仅仅用这种方法进行的。串联质谱与数据库搜索和标准的合作都需要绝对的代谢物鉴定。 代谢组学的重要组成部分是化合物的鉴定。虽然在这里详细讨论不是，质量控制的样品用LC-MS分析。多个样品制备空白和空白仪器制备用作背景减法，减少误报率从污染物，从而导致更可靠的代谢产物命中。以下这个步骤中，“分子功能”的数目根据M / Z，滞留时间，同位素比率，并加合物，以产生实际的化合物的列表被分组在一起。虽然化合物的名单被大大降低了，其结果是更加可靠的，因为它们不是基于从同一化合物的多个加合物。完整的方法是全面的，并允许isolatioÑ ​​疏水代谢产物如中性脂质，磷脂，脂肪酸，甘油三酯和甾族化合物，同时还分离亲水类中的水相，它的类二十烷酸，糖，黄酮类，与氨基酸已被确定12，26。

生物反应生成的代谢产物如细胞过程的最终产品。代谢组学是所有存在的有机体作为这些处理的结果的化合物的集合。它提供了细胞的生理学的图片，并反映生物体的响应外部或内部的刺激的1，2。这种刺激可能是环境，毒理学，药理学，营养，激素，或相关疾病。许多代谢组学的应用有，目前正在研究的研究人员，其中包括生物标志物的发现3，营养学4，食品科学5，和药物测试6。不管应用程序，不同的数据，污染和假阳性存在需要减少或优选地除去。在生物标志物的发现或确定控件和疾病组之间的差异，或者对调查对象的特效药物，生物f的情况下，LUID选择基于所提出的问题和代谢物的种类正在调查7。举例来说，如果学习的吸入药物对哮喘患者的肺部的直接影响，那么样品前探索支气管肺泡灌洗液（BALF）的代谢物和给药后会优先。为了确保观察到的差异是由于实际的生物变异，而不是不正当的样品制备技术，标准化和一致的实验室协议是必不可少的8。样品信息必须仔细记录，以确保变量，如生物流体，动物品系，采样时间，受年龄，性别，等等，都被认为并计入研究9。此外，为了减少污染或假阳性的可能性，建议该溶剂空白和空白仪器进行分析10。 对于这个协议中，术语“代谢吸住“将被用于指代所确定的实际的化合物。使用供应商的软件，初始峰值的发现算法是用来检测质谱峰。这些峰是基于质荷比（m / z）的比率和保留时间对齐。第二个算法，然后用于多种功能结合成一个单一的化合物。这包括这样一些特性如钠，钾或铵加成物中的正电离模式，和氯在​​负离子模式。在软件的其他选项包括功能，如二聚体和其他加合物。使用葡萄糖作为一个例子，与在181.0707米/ Z（M + H）的峰，198.0972米/ Z（M + NH 4），并203.05261米/ Z（M + Na）的，将有对应于相同的三个峰使用第一种算法复合。然而，当所述第二算法，该算法是基于分子式，应用这三种加合物变得组合在一起产生一种化合物。 代谢物可在SAMP造成干扰莱由于本发明化合物的复杂性。数以千计的一个样本的原因代谢物的存在信号特别的低丰度代谢产物抑制。样品净化去除干扰蛋白质和后续分离成多个部分降低了样品从而改善峰分离，提高分辨率，减少代谢产物共流出的复杂性。因此，样品净化和化合物的改进的分离是必要的。它已被证明单靠蛋白质沉淀，即使使用不同极性的溶剂，能不能解决这个问题，11，12。然而，由一强的有机溶剂如甲基叔丁基醚结合随后的分级步骤中，代谢产物覆盖率增加。使用合并的MTBE萃取溶剂Yang 等 12报道的增加的代谢物由1851或2073，用甲醇或单独的甲醇-乙醇沉淀分别以3806代谢物随后通过固相萃取（SPE）的步骤。减少代谢产物的重叠，提高了峰的分离度，并增加大量的代谢产物，观察用这种方法。 从非代谢产物，例如聚合物，污染可导致从样品采集，溶剂或仪器的噪声，并可能导致潜在的显著代谢物的信号抑制。建议技术员（S）和那些谁收集的样品之前，样品制备一直使用同一品牌，类型和样品收集瓶，移液器吸头和样品的采集和制备过程中使用的任何其他管大小。这使得数据分析人员完全有信心，所观察到的变化是真实的，而不是由于从其他来源背景的差异。治疗效力，疾病组和对照组，或者任何其他代谢分析之间的变化随后可以与增加的信心的影响。 冰毒外径这里讨论集中于可应用于血浆，支气管肺泡灌洗液或脑脊液（CSF）样品进行非靶向代谢物组学谱用于液相色谱-质谱（LCMS）分析基于组合的样品制备方法13-15。两个液相色谱（LC）和超高效液相色谱（UPLC）分离技术可以耦合到MS的此过程之后进行。进行代谢研究，许多研究者使用任一种蛋白质沉淀技术和/或液-液萃取技术16，17。在我们的研究中，这导致被检测到较少的代谢产物。这里12所述的方法，使产生更多数量的代谢产物的检测和鉴定，覆盖范围更广的代谢组。此增加是由于所引起的代谢物类的预先分离的样品和降低基体效应的较高的纯度。 初始普罗特用冷的甲醇（MeOH）纯化以除去样品中的蛋白质进行EIN沉淀步骤。液 - 液萃取（LLE）用甲基叔丁基醚（MTBE）和水用于分隔所述亲水性和疏水性的化合物。脂肪酸，中性脂质，磷脂和 - 然后固相萃取（SPE）的疏水层的疏水性化合物分离成三个等级上被执行。疏水性的级分重新溶解在100％甲醇中，而亲水部分被重新溶解在水中的5％乙腈。固相萃取（SPE）步骤提供通过减少共洗脱化合物否则会出现数量的信心，结果一个附加水平一直未得到执行的分离步骤。

<table border="0" cellpadding="0" cellspacing="0" width="628">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:167px;">Acetonitrile</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">A955-4</td>
			<td style="width:183px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:167px;">Methanol</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">L-6815</td>
			<td style="width:183px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:167px;">Chloroform</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">C606-1</td>
			<td style="width:183px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:167px;">Hexane</td>
			<td style="width:163px;">Sigma Aldrich</td>
			<td style="width:119px;">34859</td>
			<td style="width:183px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:167px;">Acetic acid</td>
			<td style="width:163px;">Sigma Aldrich</td>
			<td style="width:119px;">49199-50ML-F</td>
			<td style="width:183px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:167px;">Methyl tert-butyl ether</td>
			<td style="width:163px;">J.T. Baker</td>
			<td style="width:119px;">9042-03</td>
			<td style="width:183px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:167px;">Isopropyl alcohol</td>
			<td style="width:163px;">Sigma Aldrich</td>
			<td style="width:119px;">34965-2.5L</td>
			<td style="width:183px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:167px;">Water</td>
			<td style="width:163px;">Honeywell Burdick &#38; Jackson</td>
			<td style="width:119px;">365-4</td>
			<td style="width:183px;">-</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:167px;">OA-SYS heating system</td>
			<td style="width:163px;">Organomation Associates, Inc</td>
			<td style="width:119px;">-</td>
			<td style="width:183px;">Used to keep samples under a constant flow of nitrogen while at 35oC</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:167px;">12-position vacuum manifold</td>
			<td style="width:163px;">Phenomenex</td>
			<td style="width:119px;">-</td>
			<td style="width:183px;">-</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:167px;">Strata NH2 (55&#181;M, 70&#197;) 100mg/mL SPE cartridges</td>
			<td style="width:163px;">Phenomenex</td>
			<td style="width:119px;">8B-S009-EAK</td>
			<td style="width:183px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:167px;">Glass pipette tips</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">13-678-20C</td>
			<td style="width:183px;">Used to transfer sample to SPE column</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="2" style="height:43px;width:167px;">Plastic pipette tips</td>
			<td rowspan="2" style="width:163px;">USA Scientific</td>
			<td style="width:119px;">1182-1830</td>
			<td rowspan="2" style="width:183px;">Used when glass tips are not necessary</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:119px;">1182-8810</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:167px;">Microcentrifuge tubes</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">02-681-320</td>
			<td style="width:183px;">-</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="2" style="height:43px;width:167px;">Graduated glass pipets</td>
			<td rowspan="2" style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">13-678-27B</td>
			<td rowspan="2" style="width:183px;">Used to transfer organic solvents during sample prep</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:119px;">13-678-27E</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:167px;">Pyrex glass culture tubes</td>
			<td style="width:163px;">Corning Incorporated</td>
			<td style="width:119px;">99499-16X</td>
			<td style="width:183px;">Used to store aqueous and lipid fractions until the next step</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:167px;">Autosampler vials</td>
			<td style="width:163px;">Agilent Technologies</td>
			<td style="width:119px;">5182-0545</td>
			<td style="width:183px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:167px;">Snap cap vials for autosampler vials</td>
			<td style="width:163px;">Agilent Technologies</td>
			<td style="width:119px;">5182-0541</td>
			<td style="width:183px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:167px;">Glass inserts</td>
			<td style="width:163px;">Agilent Technologies</td>
			<td style="width:119px;">5183-2085</td>
			<td style="width:183px;">Used for small sample volumes</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:167px;">Mass Hunter Qualitative Analysis software</td>
			<td style="width:163px;">Agilent Technologies</td>
			<td style="width:119px;">Version B.06.00</td>
			<td style="width:183px;">Used to monitor retention times and pressure curves</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:167px;">Mass Hunter Quantitative Analysis software</td>
			<td style="width:163px;">Agilent Technologies</td>
			<td style="width:119px;">Version B.05.02</td>
			<td style="width:183px;">Used to analyze quality control and sample data</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:167px;">Mass Profiler Professional software</td>
			<td style="width:163px;">Agilent Technologies</td>
			<td style="width:119px;">Version B.12.50</td>
			<td style="width:183px;">Used to determine statistics, fold changes, and perform metabolite identification</td>
		</tr>
	</tbody>
</table>

multi-step preparation technique to recover multiple metabolite compound classes for in-depth and informative metabolomic analysis

代谢组学是一个新兴的领域使样品的剖面从活的生物体，以获得洞察生物过程。代谢组学的一个重要方面是样品制备据此不一致的技术产生不可靠的结果。这种技术包括蛋白质沉淀，液 - 液萃取，固相萃取作为分馏代谢成四个不同类别的一种手段。获得低丰度分子所产生的增加的敏感性提高富集，最终导致更多的自信识别的分子。这种技术已被应用到等离子，支气管肺泡灌洗液，以及与体积低至50μl的脑脊液样品。样品可以用于多个下游应用;例如，从蛋白沉淀得到的沉淀可被存储供以后分析。使用来自该步骤的上清液经历液 - 液萃取水和强有机溶剂来分离亲水性和疏水性的化合物。一旦分馏，该亲水层可以被处理以供日后分析或如果没有必要舍弃。在3固相萃取步骤以分离成脂肪酸，中性脂质和磷脂的疏水部分被进一步处理，用一系列溶剂。这使得技术人员可以灵活地选择其中的一类化合物，优选用于分析。它也有助于从化学类存在的一些知识更为可靠的代谢物鉴定。

如上所述，进行整个样品制备技术和最重要的和/或相关的方面介绍如下。亲水性和疏水性的内部标准进行了掺入混合血浆样品进行使用各种提取方法的内部标准和内源性代谢物的丰度直接比较。采用定性和定量软件液相色谱-质谱（LC-MS）数据进行分析，并导致优异的恢复和两个内源性化合物和内标分离， 图1展示了MTBE-SPE方法中提取两个脂质标准的有效性（A）和内源性化合物（B）。 总体而言，较好的提取和覆盖范围的代谢物与其它方法相比，如甲醇萃取，或&#39;MTBE只有&#39;提取得到时数ø采用定性和定量软件下LC-MS分析F特点进行了比较。例如，只用甲醇萃取，变化为肌酸酐-D 3为15.2％。然而，用MTBE液液萃取，将其降低到1.04％的CV。用甲基叔丁基醚，类脂和水的化合物的可重复性分别为&lt;分别为8％和&lt;5％，而较简单的甲醇萃取而导致的29％分别为脂质和水的化合物更大的变化和15％。用于监测脂质回收率的内部标准-睾甾酮-D 2，C17神经酰胺，15点00的PC和PE 17时00分分别增加了26％，200％，100％，400％，比单独使用甲醇。共检测到脂肪酸内部标准和磷脂酰胆碱和phosphotidylethanolamine内源性代谢物类似的增长。分别要么没有检测到其他的内源性代谢物，如鞘氨醇，神经酰胺，甘油二酯，甘油三酯，胆固醇，鞘磷脂和使用甲醇或者在可忽略的水平进行检测。然而，这些内源性脂质，用甲基叔丁基醚提取很容易被发现。 在我们的分析的比较标准协议，在取得了以下结果：甲醇沉淀独自导致了1851代谢物，甲醇 - 乙醇沉淀法得到2073的代谢物，甲基叔丁基醚用液 - 液萃取得到3125，和甲基叔丁基醚与液 - 液和固相萃取回收3,806代谢产物。因此，这种方法会导致更大的代谢物被提取，由于减少离子抑制和之前的LC-MS样品更干净，最有可能的。 图2展示了在疏水代谢产物分离成各自的化学类更有信心代谢物鉴定的效率。有在以下的SPE的3脂质部分中确定的化合物的最小重叠。为了支持， 图3显示了的内部标准，这表明内标法的洗脱在与它们的化学类馏分的回收。 质量控制样品用于评估样品制备的质量，以确定何时需要进行大样本集分析的多天的任何批次的影响，并监测仪器的重复性。色谱图进行检查，以确保尖刺入标准是大于90％，回收到小于±3ppm的和保留时间小于±5％窗口的质量误差。如果这些条件得不到满足，结果被丢弃，样品重新分析。中的批次的影响，从而在保留的移位是观察一个批次的情况下，数据分析软件可以校正这一点。将预先制备的一批收集的血浆样品进行样品制备。的馏分，然后分等分入自动进样器小瓶中并储存在-80℃下，在监测仪器条件使用蒸发散遍及每个样品分析， 表1示出了从这些尖峰在标准的结果。脂肪酸负电离模式的部分（数据在表中未示出）没有被用于分析，因为在尖峰在标准的QC样品的％CV为10％以上。因此，对于该部分的数据集被丢弃和仪器检查和维护。 表2示出了从下面的样品制备和仪器一式三份注射三种不同天内源性代谢物的样品中的结果。内源性代谢物的样品制备质控样品都是重复性的，标志着样品制备的强度以及仪器注入的重现性。 当没有正确地遵循样品制备步骤，但是，不可靠的和不一致的结果，得到图4示出了结果，当该方法的蛋白质沉淀步骤不遵循所概述。三大运营商中，A，B和C上执行收集的血浆样品相同的样品制备过程。运营商A，而不是按照实验方案吸取所需的上清液量，而不是用移液管&gt;第1毫升两种洗涤与一些颗粒。这不仅导致了许多用于该馏分误报更高，反而增加了数据的可变性。 的数据的再现性色谱可以看出，在图7。汇集血浆样品用蛋白质沉淀，液-液萃取和固相萃取在本协议中所述制备一式三份在不同的日子。使用在协议的第7条所述的色谱分离各个级分进行分析。然后将样品一式三份注射到LC-MS来评估仪器和样品制备的重现性。这种一致的重叠表明双方加强高次的在三个不同的日子时制备样品的制备，以及在制造可再现的结果的色谱法的强度的再现性。在化学噪声增加时观察到的脂肪酸馏分的负电离模式。这可能会出现由于在LC-MS溶剂和污染物可能导致不一致的定量代谢物组的结果。因此，这之前，9​​分钟只洗脱代谢物进行了分析。 当运行长的工作列表，仪器的灵敏度和变化的缓冲液浓度的损失可以发生在时间从而降低信号强度和保留时间漂移。如果保留时间重叠变化小于5％，且信号强度变化小于10％时，该数据仍然在标准实验室限制。分析软件可以被用于对齐和归一化数据，以校正仪器和保留时间漂移。但是，如果变化是拉尔GE，那么原因已被确定。一旦这被整流，样品可以被重新分析。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51670/51670fig1highres.jpg" width="500" /> 图1。脂质内标（A）和内源性代谢物（B）的下列提取和反相色谱（RPC）12的丰度 。萃取进行和所得样品使用RPC分离和在正和负电离模式中使用LC-MS分析。这个数字已经被修改Yang 等 ， 色谱1300，217-226（2013年）。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51670/51670fig2highres.jpg" width="500" /> 图2：甲基叔丁基醚-SPE的比较分数 12。在每个FR确定的代谢物动作进行比较的制备的SPE部分期间，以确定重叠量。在维恩图中的数字反映了在每个级分中检测的代谢物的数量。这里次要重叠的三个部分，即在固相萃取中成功提取的化合物和代谢类分离中观察到。这个数字已经被修改Yang 等 ， 色谱1300，217-226（2013年）。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51670/51670fig3highres.jpg" width="500" /> 图3为内标使用甲基叔丁基醚-SPE法 12 级的恢复 。萃取进行和所得样品使用RPC分离并如文中所述使用LCMS在正和负模式进行分析。这个数字已经被修改Yang 等 ， 色谱一1300，217-226（2013）。&lt;/ P&gt; <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51670/51670fig4highres.jpg" width="500" /> 图4。从结果编制三大运营商的沉淀部分 。三个样品制备运营商A，B和C进行对收集的血浆样品相同的蛋白质制备步骤。在维恩图中的数字反映了由每个操作者检测到的代谢物的数量。运营商B和C用移液管每次样品制备协议所要求的体积，而运营商A移液管整个上清液和一些沉淀，导致超过500多种代谢物，其中大部分是用于该特定部分的误报。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51670/51670fig5highres.jpg" width="500" /> 在蛋白质沉淀步骤如图5所示。形成蛋白质沉淀的&lt;/强&gt;（A）的 100微升血浆前的样品制备，加入冰冷的甲醇中后（B）的等离子体;在0℃下18000×离心15分钟后，形成在管的底部（C）的蛋白沉淀克<img alt="图6" fo:content-width="5in" src="/files/ftp_upload/51670/51670fig6highres.jpg" width="500" /> 在液-液萃取（LLE）步骤见图6。分离的亲水性和疏水层 。作为有机溶剂的甲基叔丁基醚（MTBE）和水进行分离，亲水性和疏水性的代谢产物。甲基叔丁基醚层溶解非极性化合物和水层溶解极性化合物（A）的血浆上清液后蛋白去除;在氮气下干燥后（B）的等离子体;另外的MTBE后（C）的血浆; <str<html>翁&gt;（D）加水至血浆和甲基叔丁基醚;离心后形成的（E）的甲基叔丁基醚，水层（F）去除顶部的MTBE层（G）的主要MTBE去除后剩余的亲水层。 <img alt="图7" fo:content-width="5in" src="/files/ftp_upload/51670/51670fig7highres.jpg" width="500" /> 图7。从所选数据集。样品制备三个独立的汇集血浆QC样品，每个样品进行组分的色谱图中一式三份注射到LC-MS仪器。代表的是使用的方法协议第7条指示的LC-MS参数获得的血浆样品的总离子色谱图。的其它相关生物液体色谱表示会有所不同，因为在代谢成分的差异。K“&gt;请点击这里查看该图的放大版本。 <table border="0" cellpadding="0" cellspacing="0" width="485"><colgroup><col /><col /><col /><col /><col /><col /></colgroup><tbody><tr height="42"><td height="42" style="height:42px;width:85px;">分数</td><td style="width:72px;">电离模式</td><td style="width:127px;">内标</td><td style="width:23px;"> Ñ </td><td style="width:103px;">平均峰值区</td><td style="width:77px;">峰面积的CV％ </td></tr><tr height="21"><td height="21" style="height:21px;">水</td><td>正</td><td>肌酐-D 3 </td><td> 31 </td><td> 2217311 </td><td> 3.8％ </td></tr><tr height="21"><td height="21" style="height:21px;">中性脂质</td><td>正</td><td>甘油三酯-D 5 </td><td> 31 </td><td> 4837032 </td><td> 9.9％ </td></tr><tr height="21"><td height="21" style="height:21px;"></t开发&gt; <td></td><td> C17神经酰胺</td><td> 31 </td><td> 12736707 </td><td> 7.9％ </td></tr><tr height="21"><td height="21" style="height:21px;">磷脂</td><td>正</td><td> 15:0的PC </td><td> 32 </td><td> 1248929 </td><td> 9.3％ </td></tr><tr height="21"><td height="21" style="height:21px;"></td><td></td><td> 17:0 PE </td><td> 32 </td><td> 517234 </td><td> 7.9％ </td></tr></tbody></table> 表1。质量控制结果从尖刺的内部标准 。从肺气肿模型小鼠的数据集收集的血浆样品进行分析以监测此多周的研究，每天仪器条件。定量分析软件来确定的内部标准，（正的仪器QC注射=数目）的峰面积。 <table border="0" cellpadding="0" cellspacing="0" width="555"><colgroup><col /><col /><col /><col /><col /></colgroup><tbody><tr height="42"><td height="42" style="height:42px;width:85px;">分数</td><td style="width:76px;">电离模式</td><td style="width:212px;">内源性代谢物</td><td style="width:103px;">平均峰值区</td><td style="width:80px;">峰面积的CV％ </td></tr><tr height="21"><td height="21" style="height:21px;">水</td><td>正</td><td>肌酐</td><td> 2554574 </td><td> 2.3％ </td></tr><tr height="21"><td height="21" style="height:21px;"></td><td></td><td>缬氨酸</td><td> 3712151 </td><td> 3.3％ </td></tr><tr height="21"><td height="21" style="height:21px;"></td><td></td><td>葡萄糖</td><td> 2669190 </td><td> 6.9％ </td></tr><tr height="21"><td height="21" style="height:21px;">中性脂质</td><td>正</td><td> 3 Dehydrosphinganine </td><td> 226644 </td><td> 3.9％ </td></tr><tr height="21"><td height="21" style="height:21px;"></td><td></td><t开发&gt; DG（16:0 / 16点01分/ 0:0） </td><td> 11301 </td><td> 8.2％ </td></tr><tr height="21"><td height="21" style="height:21px;"></td><td></td><td> DG（P-14：0/18：1） </td><td> 364119 </td><td> 1.9％ </td></tr><tr height="21"><td height="21" style="height:21px;">磷脂</td><td>正</td><td> PC（24:0 / 0:0） </td><td> 27599 </td><td> 0.9％ </td></tr><tr height="21"><td height="21" style="height:21px;"></td><td></td><td> PC16：0/22：6） </td><td> 2873326 </td><td> 4.5％ </td></tr><tr height="21"><td height="21" style="height:21px;"></td><td></td><td> PI（16:0 / 18:1） </td><td> 112998 </td><td> 4.4％ </td></tr><tr height="21"><td height="21" style="height:21px;">脂肪酸</td><td>正</td><td> 10 - 氧代-5,8 - 癸二烯</td><td> 1363284 </td><td> 2.3％ </td></tr><tr height="21"><td height="21" style="height:21px;"></td><td></td><td> 16 - 氧代 - 十七烷酸</td><td> 83700 </td><t开发&gt; 2.9％ </td></tr><tr height="21"><td height="21" style="height:21px;"></td><td></td><td> 2 - 甲基戊酸</td><td> 285782 </td><td> 5.7％ </td></tr><tr height="21"><td height="21" style="height:21px;">脂肪酸</td><td>负</td><td> 10 - 羟基-8 - 十八碳烯酸</td><td> 10042 </td><td> 4.9％ </td></tr><tr height="21"><td height="21" style="height:21px;"></td><td></td><td> （R） - laballenic酸</td><td> 173929 </td><td> 6.5％ </td></tr><tr height="21"><td height="21" style="height:21px;"></td><td></td><td> 2 - 酮基戊酸</td><td> 35488 </td><td> 6.0％ </td></tr></tbody></table> 表2。质量控制结果从内源性代谢物。从人类疾病数据集收集的血浆样品进行分析以监测在不同天的样品制备重现性。样品制备一式三份三天，（N = 9准备QC注射）。

Watch this Scientific Journal Video about Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis at JoVE.com

Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis

结果在代谢组学实验的可靠性取决于样品制备的有效性和可重复性。描述的是一个严格和深入的方法，使从生物体液提取的代谢物与随后分析多达数千种化合物中，或感兴趣的只是该化合物类的选项。

多步制备技术，以恢复多个代谢化合物的种类进行深入和信息化代谢组学分析

Metabolomics is an emerging field which enables profiling of samples from living organisms in order to obtain insight into biological processes. A vital ...

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JoVE Journal

Bioengineering

32.2K 次观看.  National Jewish Health. 结果在代谢组学实验的可靠性取决于样品制备的有效性和可重复性。描述的是一个严格和深入的方法，使从生物体液提取的代谢物与随后分析多达数千种化合物中，或感兴趣的只是该化合物类的选项。 

视频: Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)7, 5, 9, developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)1, 4, 8, vascular networks (India ink)1, 4, and cell body distribution (Nissl)3. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain6, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research10; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. 
In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.

The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.

刀口扫描显微镜的样品制备，图像和分析协议

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at ...

科研

生物工程

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 ...

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

一种微流体装置研究多个不同的菌株

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

含有金纳米棒蜂窝车辆的制备及光声分析

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

TAPE: A Biodegradable Hemostatic Glue Inspired by a Ubiquitous Compound in Plants for Surgical Application

This video describes the simplest protocol for preparing biodegradable surgical glue that has an effective hemostatic ability and greater water-resistant adhesion strength than commercial tissue adhesives. Medical adhesives have attracted great attention as potential alternative tools to sutures and staples due to their convenience in usage with minimal invasiveness. Although there are several protocols for developing tissue adhesives including those commercially available such as fibrin glues and cyanoacrylate-based materials, mostly they require a series of chemical syntheses of organic molecules, or complicated protein-purification methods, in the case of bio-driven materials (i.e., fibrin glue). Also, the development of surgical glues exhibiting high adhesive properties while maintaining biodegradability is still a challenge due to difficulties in achieving good performance in the wet environment of the body. We illustrate a new method to prepare a medical glue, known as TAPE, by the weight-based separation of a water-immiscible supramolecular aggregate formed after a physical mixing of a plant-derived, wet-resistant adhesive molecule,&#160;Tannic Acid (TA), and a well-known biopolymer, Poly(Ethylene) glycol (PEG). With our approach, TAPE shows high adhesion strength, which is 2.5-fold more than commercial fibrin glue in the presence of water. Furthermore, TAPE is biodegradable in physiological conditions and can be used as a potent hemostatic glue against tissue bleeding. We expect the widespread use of TAPE in a variety of medical settings and drug delivery applications, such as polymers for muco-adhesion, drug depots, and others.

We describe the simplest protocol to prepare biodegradable medical glue that has an effective hemostatic ability. TAPE is a water-immiscible supramolecular aggregate prepared by mixing of tannic acid, a ubiquitous compound found in plants, and poly(ethylene) glycol, yielding a 2.5 times greater water-resistant adhesion compared with commercial fibrin glue.

TAPE：一种生物可降解止血胶由无处不在的复方植物外科应用的启发

This video describes the simplest protocol for preparing biodegradable surgical glue that has an effective hemostatic ability and greater water-resistant ...

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular level in the chemical and biomedical sciences. Although commercial SIM systems are available, systems that are custom designed in the laboratory can outperform commercial systems, the latter typically designed for ease of use and general purpose applications, both in terms of imaging fidelity and speed. This article presents an in-depth guide to building a SIM system that uses total internal reflection (TIR) illumination and is capable of imaging at up to 10 Hz in three colors at a resolution reaching 100 nm. Due to the combination of SIM and TIRF, the system provides better image contrast than rival technologies. To achieve these specifications, several optical elements are used to enable automated control over the polarization state and spatial structure of the illumination light for all available excitation wavelengths. Full details on hardware implementation and control are given to achieve synchronization between excitation light pattern generation, wavelength, polarization state, and camera control with an emphasis on achieving maximum acquisition frame rate. A step-by-step protocol for system alignment and calibration is presented and the achievable resolution improvement is validated on ideal test samples. The capability for video-rate super-resolution imaging is demonstrated with living cells.

This article provides an in depth guide for the assembly and operation of a structured illumination microscope operating with total internal reflection fluorescence illumination (TIRF-SIM) to image dynamic biological processes with optical super-resolution in multiple colors.

指南结构照明显微镜TIRF在高速，多种颜色

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

多级可变高度的光刻多层带瓣微流体装置

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

毛细管阵列中多核酸的视觉检测

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

使用一个荧光显微镜进行多种成像模式

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...