The authors thank Professor H. Troy Ghashghaei from NCSU Department of Molecular Biomedical Sciences for providing the whole mouse tissue. The authors also gratefully acknowledge the financial assistance received from National Institutes of Health (R01GM087964), the W.M. Keck foundation, and North Carolina State University.

注:以下协议描述的所有执行IR-MALDESI MSI实验的必要步骤。深入可以在其他地方5,6-找到关于红外MALDESI源的优化几何形状及其与激光，阶段同步和质谱仪的信息。在这个协议中使用的动物组织样品按照机构动物护理和使用委员会（IACUC）和北卡罗莱纳州立大学的规定取得。 1.组织准备<ol><li>通过在通风橱中放置〜200毫升异戊烷在干净的烧杯干冰的次级容器内制备异戊烷/干冰浴。在任何时候都处理异戊烷/干冰浴和组织时，使用防护手套和护目镜。 <ol><li>通过安乐死过量溴乙醇2日龄整个新生小鼠小狗（7.5毫克/克体重），然后冻结组织中的异戊烷/干冰浴保持组织结构。使用一个预清洁编对镊子来放置，并从异戊烷/干冰浴移开鼠标小狗中cryomold。在-80℃直至分析存储快速冷冻组织。 </li></ol></li><li>使用防护手套，应用最佳切割温度（OCT）安装介质的层至40mm的低温恒温器试样圆盘。冷冻整个鼠标轻轻放置到华侨城涂层试样架坚持鼠标光盘在所需方向进行切片。 注意:OCT用于仅粘附的组织，以在试样圆盘用于切片。不要完全嵌入组织中OCT和华侨城是已知有MSI分析不利影响避免过剩。其他媒体，例如明胶，可用于冷冻嵌入组织切片它9之前。 </li><li>放在珀尔帖样品架光盘（与华侨城和组织切片）的低温恒温器，按佩尔蒂埃电源按钮。等待10分钟样品来热平衡。 </li><li>放置标本盘（与组织安装在它）该盘片保持器内，并面对组织向下到用于分析期望的平面。切片组织成使用容纳在低温恒温器10内的旋转式切片机在-20℃下所需厚度的部分。 注意:对于全身分析，切片组织成25微米厚的部分，以保持组织完整性。为更小的区域（ 例如 ，肝，脑，肾），切片组织成10微米厚的切片。 <ol><li>使用防倾玻璃板，以防止切片组织切片从滚动。使用低温恒温器真空软管和刷子去除不需要的组织碎片。一旦组织切片，用刀片护罩覆盖锋利的切片机刀片，以免造成人身伤害。 </li></ol></li><li>对试样板东方鼠部分和通过使滑动尽可能靠近于组织部分解冻贴装到预清洗的玻璃显微镜玻片，而不触及它10。 注:对于定量MSI experiments，涂层用自动气动喷雾器安装所述组织之前，与内部标准的幻灯片，和解冻贴装上涂布的载玻片11上的组织切片。 </li><li>删除习惯使用刀片喷射器切片组织切片机刀片，并安全地丢弃在适当的"锐利废物"容器中的刀片。 </li><li>保持低温恒温器内部的载玻片，直到步骤3.2，以保持组织的冷冻保存。 </li></ol> 2.红外MALDESI准备/校准<ol><li>打开中红外激光发射计算机上的激光控制中的应用。选择使用由制造商提供的激光控制应用所需的波长。红外MALDESI实验在2940纳米下进行。 注:最近的实验研究中红外的激光波长的依赖和冰矩阵这似乎是具体 ​​的分析指出8差异。 </li><li>打开雄鹿E控制器，启动自定义用户控制程序（RASTIR），和校准使用home键的舞台上的地位。 </li><li>准备电的解决方案。通常情况下，使用以50:50的比例（体积/体积）甲醇/水含0.2％甲酸在正离子模式电喷雾溶剂。根据实验选择电溶剂组合物。例如，使用5毫米氢氧化铵在负离子模式成像应用电改性剂。 注意:对于极性转换红外MALDESI微星，使用1mM的乙酸作为改性剂。这种改性剂被发现最适合于在两个正，负离子模式12获得稳定的和可重复的信号。 </li><li>填充1ml注射器具有电溶剂，并冲洗用新溶剂中的二氧化硅毛细管。 </li><li>使用源的参数，如:ESI-光点距离对齐ESI发射极上轴与MS入口，点进距离，样品的高度，并溶剂流速已使用统计DO优化<html> E代表组织切片6的分析。注:参见图1示意描绘这些参数的优化值组织切片的MSI。 </li><li>启动电和评价通过监测总离子电流（TIC）其中，在这一点上，仅由环境化合物其稳定性（&gt; 10分钟）。典型地，&lt;10％以上10〜15分钟的TIC变化是稳定的良好指示。 </li></ol><img alt="图1" src="/files/ftp_upload/53942/53942fig1.jpg" /> 图1. IR-MALDESI原理和参数。（A）IR-MALDESI源设置（不按比例）和可调节参数的示意图。 （ 二 ）优化参数值组织切片成像。 <a href="https://www.jove.com/files/ftp_upload/53942/53942fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 冰阵标题"&gt; 3。沉积<ol><li>注意:将样品在舞台前，关闭电。 </li><li>放置解冻上的组织到摄像机视野内的红外MALDESI样品板。 </li><li>确保手是明确ESI发射器并重新启动电。 </li><li>关闭红外MALDESI源和净化机箱的访问门用干燥的氮气，以防止水凝结所述组织的表面上。一旦外壳到达内部的相对湿度&lt;3％，打开直流电源（〜12V）到珀耳帖板，其将冷却所述样品板。注意:在珀耳帖冷却阶段的冷却过程是在别处详细13讨论。 </li><li>冷却阶段-9℃，并允许上的组织，以达到热平衡（〜5-10分钟）。 </li><li>停止氮气吹扫，并且通过打开源接入门暴露组织的环境相对湿度。冰的薄层将沉积在共珀尔帖阶段的LD表面和安装组织部分由于来自空气的水凝华。 注意:如果在实验室的相对湿度低于10％-15％，将温水的烧杯中的外壳内以促进冰基质层的形成。 </li><li>形成在冰基质层后，关闭检修门，并重新启动干氮气的流动，以相对湿度降低至10±2％。调整氮气流量，以保持相对湿度在这个水平上，经验上发现的冰的形成和升华的平衡整个实验6保持冰的恒定层。 </li></ol> 4.质谱成像数据采集<ol><li>使用RASTIR GUI（ 如图2），点击"LASER位置"按钮，移动舞台分析位置。使用激光的调整二极管以确保离组织区域将被消融。火场外的组织重新激光射击祗园校准激光相对于摄像机视图所抵消。点击"激光测试火"按钮，更新激光位置。 </li><li>返回到"相机位置"，并通过将激光对准十字线的激光烧蚀点（ 图2-1）更新RASTIR激光位置。 </li><li>点击感兴趣区域（ROI）按钮的区域和调整ROI的大小和位置的组织切片被分析（ 图2-2）。输入相应的实验参数，如X和Y方向（单位mm）（ 图2-3）步长，并命名在"项目名称"框（ 图2-4），MSI文件。 <ol><li>当绘制ROI，包括了组织，充当控制的一部分。使用高峰采摘这一关，组织切片（步骤5.4）。 注意:在组织中的激光的解吸直径（点尺寸）大约为150微米;然而，更高的空间分辨率可以通过USI获得纳克过采样方法14,15。 </li></ol></li><li>选择从下拉框中，每像素的激光脉冲（整数值）的数目，以及激光触发器和质谱仪信号采集（ 图2-5）之间的延迟的适当的激光频率。通常使用每像素两个激光脉冲在20赫兹到在IR-MALDESI微星实验完全解吸材料，具有10毫秒的延迟时间，以允许产生到达质量分析器测量5离子。 注:选择最高的重复率使激光能够在操作。这是最近表明，使用较高的重复率将提高分析物探测16。 </li></ol><img alt="图2" src="/files/ftp_upload/53942/53942fig2.jpg" /> 图2.用户界面IR-MALDESI MSI操作。在RASTIR扫描控制程序的截图呈现。对于PERF的步骤orming一个微星实验是:（1）定位的激光点，（2）绘制的ROI，（3）在选择阶段步长大小（单位mm），（4）赋予的名称到该文件，（5）选择正确的数每个像素脉冲与所需的重复率，（6）检查列表成像和MS设置沿。 <a href="https://www.jove.com/files/ftp_upload/53942/53942fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="5"><li>选择质谱仪的参数，如电离模式下，电压，溶剂流速，毛细管温度，并在质谱仪软件喷射时间。参考表1对在全身的IR-MALDESI微星使用的参数的一个例子。 注意:由于在生物样品和缺乏微星分析分离方法的复杂性，对IR-MALDESI源耦合到一个高分辨力和高的质量准确度的仪器。采集方法可以加载analysES如平行反应监测（PRM）7或极性切换MSI 12。 </li><li>一旦所有参数（激光，舞台，和质谱仪）已被选定，将在MS同步"握手"模式，并启动MS采集。 </li><li>使用RASTIR成像软件，验证所有步骤清单（ 图2-6）通过检查框完成，加载程序。一旦程序被加载，"运行"按钮将变为可用。按Run启动MSI信号采集。 </li><li>一旦成像实验完成后，停止质谱仪采集并放置在待机状态下在仪器。关闭氮气吹扫到机箱，关掉电源，珀耳帖板，并关闭级控制器和激光。 </li><li>打开检修门，从源头机箱内取出电发射头，并使用防护手套删除扩展加热的金属质谱入口。注意:扩展<html>金属质谱入口会很烫。 </li><li>戴手套和护目镜，清洗在15％的硝酸经超声波浴中的扩展金属毛细管入口10分钟，然后在HPLC级水10分钟，最后在HPLC级甲醇10分钟。擦干金属毛细管入口在氮气流中，并重新插入MS。注:在适当的废物容器后处置溶剂。 </li></ol><table border="0" cellpadding="0" cellspacing="0" width="432"><colgroup><col span="2" /></colgroup><tbody><tr height="22"><td dir="LTR" height="22" style="height:22px;width:216px;"> 参数 </td><td dir="LTR" style="width:216px;"> 值 </td></tr><tr height="21"><td dir="LTR" height="21" style="height:21px;width:216px;">电离模式</td><td dir="LTR" style="width:216px;">正面的</td></tr><tr height="21"><td dir="LTR" height="21" style="height:21px;width:216px;">电电压</td><html> R"的风格="WIDTH:216px;"&gt; 3.8-4.2千伏</td></tr><tr height="21"><td dir="LTR" height="21" style="height:21px;width:216px;">溶剂流速</td><td dir="LTR" style="width:216px;"> 2微升/分钟</td></tr><tr height="21"><td dir="LTR" height="21" style="height:21px;width:216px;">毛细管温度</td><td dir="LTR" style="width:216px;"> 275℃ </td></tr><tr height="21"><td dir="LTR" height="21" style="height:21px;width:216px;">扫描范围</td><td dir="LTR" style="width:216px;"> M / Z 250-1000 </td></tr><tr height="21"><td dir="LTR" height="21" style="height:21px;width:216px;">扫描类型</td><td dir="LTR" style="width:216px;">全扫描</td></tr><tr height="21"><td dir="LTR" height="21" style="height:21px;width:216px;">注射时间</td><td dir="LTR" style="width:216px;"> 110毫秒</td></tr><tr height="22"><td dir="LTR" height="22" style="height:22px;width:216px;">解析力</td>宽度:216px;"&gt; 14万</td></tr></tbody></table>在全身IR-MALDESI微星采用表1.仪器参数。 5.数据分析<ol><li>由仪器生成的原始数据文件转换为数据格式，如mzXML 17或18 imzML使用免费软件如MSConvert 17和imzML转换器19。 </li><li>开始MSiReader V1.0，高分辨力MSI数据20的分析制定，有专门的处理计算机上的开源软件。加载映像文件到MSiReader。对于MSiReader的用户界面和它的一些内置的功能，参见图3。 </li><li>通过输入其M / Z生成的目标分析物离子地图，选择百万分之适当的M / Z窗口（PPM）或汤姆森（TH）（ 图3-1）。对于高分辨率（RP）仪器选择在低ppm范围内的一个窗口。 注:将合适的M / Z窗口取决于仪器即IR-MALDESI源耦合到的RP和质量的测量精度（MMA）。 </li><li>进一步解释使用内置的功能，如插补（ 图3-2），标准化（ 图3-3），光学图像叠加（ 图3-4），和峰值采摘（ 图3-5）20的数据。 </li></ol><img alt="图3" src="/files/ftp_upload/53942/53942fig3.jpg" /> 图3. MSiReader的用户界面; V1.0 20。一旦一个文件被加载到软件中，感兴趣分析物的离子映射由（1）输入ppm或钍的M / Z和公差显示。还可以执行诸如（2）内插或（3）归一化进一步分析。组织的光学图像也可以导入并叠加离子的地图（4）为更好的可视化。对于不相关的分析峰的拾取功能（5）可用于通过选择组织（品红色线）的区域和参考区域外组织（绿盒）提取组织特异性的峰值。 <a href="https://www.jove.com/files/ftp_upload/53942/53942fig3large.jpg" target="_blank">请点击此处查看大图这个数字。</a> <ol start="5"><li>使用峰拾取函数生成使用用户定义的标准组织特异性m / z值的列表。注:PeakFinder算法使用两个区域平均质谱差异。然后，这些m / z值可以搜索针对代谢物数据库如METLIN 21或脂质MAPS 22 <ol><li>参考图3为选择一个询问组织区域（品红多边形ROI），并关断组织的参考区域（绿色多边形ROI）的一个例子。 </li></ol></li></ol>

<ol>
	<li>Mcdonnell, L. A., Heeren, R. 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=Imaging+Mass+Spectrometry.">Imaging Mass Spectrometry.</a> Mass Spectrom. Rev. 26, 606-643 (2007).</li><li>Chughtai, K., Heeren, R. 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=Mass+spectrometric+imaging+for+biomedical+tissue+analysis.">Mass spectrometric imaging for biomedical tissue analysis.</a> Chem. Rev. 110 (5), 3237-3277 (2010).</li><li>Robichaud, G., Barry, J. A., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atmospheric+Pressure+Mass+Spectrometry+Imaging.">Atmospheric Pressure Mass Spectrometry Imaging.</a> Encycl. Anal. Chem. , (2014).</li><li>Sampson, J. S., Hawkridge, A. M., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+detection+of+multiply-charged+peptides+and+proteins+by+matrix-assisted+laser+desorption+electrospray+ionization+(MALDESI)+Fourier+transform+ion+cyclotron+resonance+mass+spectrometry.">Generation and detection of multiply-charged peptides and proteins by matrix-assisted laser desorption electrospray ionization (MALDESI) Fourier transform ion cyclotron resonance mass spectrometry.</a> J. Am. Soc. Mass Spectrom. 17 (12), 1712-1716 (2006).</li><li>Robichaud, G., Barry, J. A., Garrard, K. P., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infrared+matrix-assisted+laser+desorption+electrospray+ionization+(IR-MALDESI)+imaging+source+coupled+to+a+FT-ICR+mass+spectrometer.">Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) imaging source coupled to a FT-ICR mass spectrometer.</a> J. Am. Soc. Mass Spectrom. 24 (1), 92-100 (2013).</li><li>Robichaud, G., Barry, J. A., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IR-MALDESI+Mass+Spectrometry+Imaging+of+Biological+Tissue+Sections+Using+Ice+as+a+Matrix.">IR-MALDESI Mass Spectrometry Imaging of Biological Tissue Sections Using Ice as a Matrix.</a> J. Am. Soc. Mass Spectrom. 25 (3), 319-328 (2014).</li><li>Barry, J. A., 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=Mapping+Antiretroviral+Drugs+in+Tissue+by+IR-MALDESI+MSI+Coupled+to+the+Q+Exactive+and+Comparison+with+LC-MS/MS+SRM+Assay.">Mapping Antiretroviral Drugs in Tissue by IR-MALDESI MSI Coupled to the Q Exactive and Comparison with LC-MS/MS SRM Assay.</a> J. Am. Soc. Mass Spectrom. 25 (12), 2038-2047 (2014).</li><li>Rosen, E. P., Bokhart, M. T., Ghashghaei, H. T., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+Desorption+Conditions+on+Analyte+Sensitivity+and+Internal+Energy+in+Discrete+Tissue+or+Whole+Body+Imaging+by+IR-MALDESI.">Influence of Desorption Conditions on Analyte Sensitivity and Internal Energy in Discrete Tissue or Whole Body Imaging by IR-MALDESI.</a> J. Am. Soc. Mass Spectrom. 26, 899-910 (2015).</li><li>Nelson, K. A., Daniels, G. J., Fournie, J. W., Hemmer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+whole-body+zebrafish+sectioning+methods+for+mass+spectrometry+imaging.">Optimization of whole-body zebrafish sectioning methods for mass spectrometry imaging.</a> J. Biomol. Tech. 24 (3), 119-127 (2013).</li><li>Park, J. J., Cunningham, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thin+sectioning+of+slice+preparations+for+immunohistochemistry.">Thin sectioning of slice preparations for immunohistochemistry.</a> J. Vis. Exp. (3), e194 (2007).</li><li>Bokhart, M. T., Rosen, E., Thompson, C., Sykes, C., Kashuba, A. D. M., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+mass+spectrometry+imaging+of+emtricitabine+in+cervical+tissue+model+using+infrared+matrix-assisted+laser+desorption+electrospray+ionization.">Quantitative mass spectrometry imaging of emtricitabine in cervical tissue model using infrared matrix-assisted laser desorption electrospray ionization.</a> Anal. Bioanal. Chem. 407 (8), 2073-2084 (2015).</li><li>Nazari, M., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarity+Switching+Mass+Spectrometry+Imaging+of+Healthy+and+Cancerous+Hen+Ovarian+Tissue+Sections+by+Infrared+Matrix-Assisted+Laser+Desorption+Electrospray+Ionization+(IR-MALDESI).">Polarity Switching Mass Spectrometry Imaging of Healthy and Cancerous Hen Ovarian Tissue Sections by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI).</a> Analyst. 141, 595-605 (2016).</li><li>Hsu, C. 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=Design+and+Application+of+a+Low-Temperature+Peltier-Cooling+Microscope.">Design and Application of a Low-Temperature Peltier-Cooling Microscope.</a> J. Pharm. Sci. 85 (1), 70-74 (1996).</li><li>Jurchen, J. C., Rubakhin, S. S., Sweedler, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI-MS+imaging+of+features+smaller+than+the+size+of+the+laser+beam.">MALDI-MS imaging of features smaller than the size of the laser beam.</a> J. Am. Soc.Mass Spectrom. 16 (10), 1654-1659 (2005).</li><li>Nazari, M., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular-level+mass+spectrometry+imaging+using+infrared+matrix-assisted+laser+desorption+electrospray+ionization+(IR-MALDESI)+by+oversampling.">Cellular-level mass spectrometry imaging using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) by oversampling.</a> Anal. Bioanal. Chem. 407 (8), 2265-2271 (2015).</li><li>Rosen, E. P., Bokhart, M. T., Nazari, M., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+C-Trap+Ion+Accumulation+Time+on+the+Detectability+of+Analytes+in+IR-MALDESI+MSI.">Influence of C-Trap Ion Accumulation Time on the Detectability of Analytes in IR-MALDESI MSI.</a> Anal. Chem. 87, 10483-10490 (2015).</li><li>Kessner, D., Chambers, M., Burke, R., Agus, D., Mallick, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ProteoWizard:+open+source+software+for+rapid+proteomics+tools+development.">ProteoWizard: open source software for rapid proteomics tools development.</a> Bioinformatics. 24 (21), 2534-2536 (2008).</li><li>Schramm, T., 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=ImzML+-+A+common+data+format+for+the+flexible+exchange+and+processing+of+mass+spectrometry+imaging+data.">ImzML - A common data format for the flexible exchange and processing of mass spectrometry imaging data.</a> J. Proteomics. 75 (16), 5106-5110 (2012).</li><li>Race, A. M., Styles, I. B., Bunch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inclusive+sharing+of+mass+spectrometry+imaging+data+requires+a+converter+for+all.">Inclusive sharing of mass spectrometry imaging data requires a converter for all.</a> J. Proteomics. 75 (16), 5111-5112 (2012).</li><li>Robichaud, G., Garrard, K. P., Barry, J. A., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MSiReader:+an+open-source+interface+to+view+and+analyze+high+resolving+power+MS+imaging+files+on+Matlab+platform.">MSiReader: an open-source interface to view and analyze high resolving power MS imaging files on Matlab platform.</a> J. Am. Soc. Mass Spectrom. 24 (5), 718-721 (2013).</li><li>Smith, C. A., O'Maille, G., 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=METLIN:+a+metabolite+mass+spectral+database.">METLIN: a metabolite mass spectral database.</a> Ther. Drug. Monit. 27 (6), 747-751 (2005).</li><li>Sud, 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=LMSD:+LIPID+MAPS+structure+database.">LMSD: LIPID MAPS structure database.</a> Nucleic Acids Res. 35, D527-D532 (2007).</li><li>Schwartz, S. A., Reyzer, M. L., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+tissue+analysis+using+matrix-assisted+laser+desorption/ionization+mass+spectrometry:+practical+aspects+of+sample+preparation.">Direct tissue analysis using matrix-assisted laser desorption/ionization mass spectrometry: practical aspects of sample preparation.</a> J. Mass Spectrom. 38 (7), 699-708 (2003).</li><li>Takai, N., Tanaka, Y., Inazawa, K., Saji, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+pharmaceutical+drug+distribution+in+multiple+organs+by+imaging+mass+spectrometry.">Quantitative analysis of pharmaceutical drug distribution in multiple organs by imaging mass spectrometry.</a> Rapid Commun. Mass Spectrom. 26 (13), 1549-1556 (2012).</li><li>Liu, J., Gingras, J., Ganley, K. P., Vismeh, R., Teffera, Y., Zhao, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-body+tissue+distribution+study+of+drugs+in+neonate+mice+using+desorption+electrospray+ionization+mass+spectrometry+imaging.">Whole-body tissue distribution study of drugs in neonate mice using desorption electrospray ionization mass spectrometry imaging.</a> Rapid Commun. Mass Spectrom. 28 (2), 185-190 (2014).</li></ol>

The authors declare no competing financial interests.

上述协议描述为执行IR-MALDESI MSI实验的关键步骤。矩阵应用程序（第3节）大约需要20分钟，这是通过升华或类似的用于MALDI微星实验的典型矩阵应用过程使用机器人喷雾器喷涂。此外，红外MALDESI不依赖于分析物的分配到基质晶体6和冰基质可普遍用于所有分析物，无论它们的质量，大小，或化学性质的。此外，利用冰的消除了在典型的MALDI实验23观察到较低的m / z范围的矩阵相...

在微探针模式质谱成像（MSI）通过在样品的表面上的离散位置的光束（激光或离子）涉及从表面样品的解吸。在每个栅格点，则产生的质谱和所获取的光谱，与从它们被收集的空间位置一起，可以用来同时映射样本内的许多分析物。此无标记耦合到灵敏度和质谱特异性成像的方式帮助微星成为质谱1,2-最快速发展的领域之一。 基质辅助激光解吸/电离（MALDI）是用于MSI分析中最常见的电离方法。然而，需要一种有机基质和MALDI的真空要求姿势上再现性，样品通量，并且可以使用该方法分析的样品的类型显著局限性。许多大气压力（AP）的IOnization方法已被开发，近年来，以规避这些限制3。这些环境电离方法允许生物样品的分析中是更接近自然状态，并简化分析前样品制备步骤的环境。基质辅助激光解吸电喷雾电离（MALDESI）是这样一种电离法4,5的一个例子。 在IR-MALDESI实验中，冰的薄层沉积在组织表面作为能量吸收基质上。中红外激光脉冲由冰基质吸收，并促进中性材料的解吸从由共振激励OH表面伸展的水模式。解吸的中立分区为正交电的带电滴，并且后电离在ESI状方式4-6。外源性冰矩阵的加法优于在组织上的内源性水单靠，因为它有助于交流算用于在不同组织区室含水量的变化，并且已显示通过〜15倍的组织成像实验7,8，以提高解吸6和提高离子丰度。 在这项工作中，我们采用了IR-MALDESI MSI引发的新生小鼠全身不同器官的代谢产物的分布。对IR-MALDESI源的可调节的参数的概况给出，并且对组织切片的成功的成像必要的步骤被证明。

<table border="0" cellpadding="0" cellspacing="0" width="963">
	<tbody>
		<tr height="69">
			<td height="69" style="height:69px;width:283px;">IR-MALDESI Source</td>
			<td style="width:188px;">Custom-made</td>
			<td style="width:171px;">N/A</td>
			<td style="width:323px;">Please refer to references 4 and 12 for an in-depth discussion of IR-MALDESI source development.</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:283px;">Q Exactive Plus&#160;</td>
			<td style="width:188px;">Thermo Scientific</td>
			<td style="width:171px;"></td>
			<td style="width:323px;">Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Water, HPLC Grade</td>
			<td style="width:188px;">Burdick &#38; Jackson&#160;</td>
			<td style="width:171px;">AH365-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Methanol, HPLC Grade</td>
			<td style="width:188px;">Burdick &#38; Jackson&#160;</td>
			<td style="width:171px;">AH230-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Formic Acid</td>
			<td style="width:188px;">Sigma Aldrich&#160;</td>
			<td style="width:171px;">56302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Tunable mid-IR Laser</td>
			<td style="width:188px;">Opotek Inc.</td>
			<td style="width:171px;">IR Opolette</td>
			<td>Tunable 2700-3100 nm IR OPO laser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Nitrogen Gas</td>
			<td style="width:188px;">Arc3 Gases</td>
			<td style="width:171px;">AG S-NI300-5.0</td>
			<td>Grade 5.0 high purity nitrogen gas cylinder (300)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Cryostat</td>
			<td style="width:188px;">Leica Biosystems</td>
			<td style="width:171px;">CM 1950</td>
			<td>Cryomicrotome</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">High Profile Microtome Blades</td>
			<td style="width:188px;">Leica Biosystems</td>
			<td>3802123</td>
			<td>Leica DB80HS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Mounting Medium (OCT)</td>
			<td style="width:188px;">Leica Biosystems</td>
			<td style="width:171px;">3801480</td>
			<td>Surgipath FSC 22 mounting medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Cryostat Specimen Disc</td>
			<td style="width:188px;">Leica Biosystems</td>
			<td style="width:171px;">14047740045</td>
			<td>40 mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Glass Microscope Slides</td>
			<td style="width:188px;">VWR</td>
			<td style="width:171px;">48312-003</td>
			<td>Frosted, selected, pre-cleaned</td>
		</tr>
	</tbody>
</table>

whole-body mass spectrometry imaging by infrared matrix-assisted laser desorption electrospray ionization (ir-maldesi)

对于质谱（MS）的环境电离源已在过去的十年中很大兴趣的主题。基质辅助激光解吸电喷雾电离（MALDESI）是这样的方法，一个例子，其中基质辅助激光解吸/电离（MALDI）（ 例如，解吸的脉冲性质）和电喷雾电离（ESI）（ 例如，软电离的特征）组合。之一的MALDESI的主要优点是其固有的多功能性。在MALDESI实验中，紫外线（UV）或红外线（IR）激光可用于共振激发内源或外源矩阵。矩阵的选择不是分析物依赖性的，并且仅取决于用于激发的激光波长。在IR-MALDESI实验中，冰的薄层沉积在样品表面作为能量吸收基质上。对IR-MALDESI源几何已经使用的试验设计（DOE），用于液体样品的分析统计设计以及生物化学优化ogical组织标本。此外，一强大的红外MALDESI成像源已被开发出来，其中，可调谐的中红外激光器用计算机控制的XY平移阶段和高的分辨能力质谱仪同步。自定义的图形用户界面（GUI）允许激光的重复频率的用户选择，每像素镜头，样品台的步长，和解吸之间的延迟和扫描源的事件的数目。红外MALDESI已在多种应用中被使用，如生物组织切片的纤维和染料和MSI的取证分析。不同的分析物，从内源性代谢物的组织切片中的外源外源物的分布可以被测量，并使用这种技术进行定量。在这个手稿介绍的协议详细描述了全身组织切片的IR-MALDESI MSI主要步骤。

在图4中呈现的图像显示在不同器官中的全身组织切片的代谢物的空间分布。使用MSiReader PeakFinder发现独特m / z值到身体的特定区域，接着通过分批处理的图像生成。图像叠加工具（ 图3-4）被用来对准所得离子地图冰基质沉积之前拍摄的光学图像。胆固醇是在所有组织类型观察到如从动物细胞膜（ 图4A）其结构的作用预期的，而?...

Watch this Scientific Journal Video about Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI at JoVE.com

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI

A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.

全身质谱成像红外基质辅助激光解吸电喷雾电离（IR-MALDESI）

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption ...

whole-body-mass-spectrometry-imaging-infrared-matrix-assisted-laser

Research

JoVE Journal

Bioengineering

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

9.4K 次观看.  North Carolina State University. A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse. 

视频: Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for sample pretreatment. The method was first described by Fenn and colleagues1 and has been applied to the detection of illicit drugs2 and explosives3-4, the characterization of skin volatiles5, and the analysis of breath6-7.
SESI ionization occurs by proton transfer reactions between the electrospray solution and the volatile analyte, and is therefore suitable for the analysis of hetero-organic molecules, just as in traditional electrospray ionization (ESI). However, unlike standard ESI, the proton transfer process of SESI occurs in the vapor phase rather than in solution (Fig. 1), and therefore SESI is best suited for detecting organic volatiles and aerosols.
We are expanding the use of SESI-MS to the detection of bacterial volatiles as a method for bacterial identification and characterization8. We have demonstrated that SESI-MS volatile fingerprinting, combined with a statistical analysis method, can be used to differentiate bacterial genera, species, and mixed cultures in a variety of growth media.8 Here we provide the steps for obtaining bacterial volatile fingerprints using SESI-MS, including the instrumental parameters that should be optimized to ensure robust bacterial identification and characterization.

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

Secondary electrospray ionization mass spectrometry (SESI-MS) enables the detection of volatile organic compounds (VOCs) without the need for any sample pretreatment. This protocol provides instructions for the rapid (within minutes) characterization of bacterial VOCs using SESI-MS.

使用辅助电喷雾质谱（SESI - MS）表征细菌挥发

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for ...

科研

生物工程

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

解吸电喷雾电离质谱成像的生物组织

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em&gt;在体内</em&gt;光学成像

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

样品制备策略的3D细胞培养模型质谱成像

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

从混合三维培养使用近红外Photoimmunotherapy技术选择性细胞消除

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

用质谱和荧光法研究非规范氨基酸和蛋白质分析的选择性压力下工程 "金" 荧光

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

斜扫描激光检眼镜 (奥斯陆) 与光学相干层析成像 (OCT) 的多模态视网膜成像

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

全身质谱成像红外基质辅助激光解吸电喷雾电离（IR-MALDESI）

摘要

探索更多视频

此视频中的章节