支持这项工作是由ARRA NSF MRI仪器开发补助金＃0923179 FMF。我们感谢水族Asberry，帕克·佩蒂特生物工程研究所和生物科学组织学核心实验室协调员，协助组织切片。

1。组织切片<ol><li>商店闪光灯冷冻，在-80℃冷冻，直到整个组织准备切片。 </li><li>允许的组织样本中的温度达到之前的冷冻切片机切片（30分钟）。设置的叶片和样品温度至-30℃。 </li><li>一旦组织中已达到的温度，处理的试样用小钳子，切正面或背面的脑的部分的大脑还有足够的安装表面（ 即 ，如果前面的大脑是最重要的，大脑后部安装卡盘上）。 <ol><li>应用〜0.5毫升最佳切削温度化合物（华侨城，组织康樱）夹头中心，使用镊子，并保持样品在华侨城的地方，直到它变硬。华侨城一旦被完全冻结，安装夹头样品架。 </li><li>用于装载样品的OCT优选的最小量（对比试样安装在一个块中的OCT是常见的做在其他组织相容逻辑程序），以尽量减少样品的污染。样品的污染，由华侨城损害到DESI和成像过程中，由于离子抑制。 </li></ol></li><li>典型的组织切片所需的MS的成像范围从12至18微米厚。节组织的建议范围内所需的厚度。解冻挂载每个切片组织超过部分室温下的显微载玻片轻轻触摸。一旦样品被安装，请小心不要触摸，因为它会改变组织切片的化学分布和组成。 </li></ol>注意:我们建议安装两个部分每张幻灯片，使用一节进行优化，和其他成像。如果部分是没有立即成像，存储滑梯-80℃的冰箱在幻灯片中，直到准备好进行分析。 <ol start="5"><li>如果分析存储部分，从冰箱中删除幻灯片，立即转移到真空干燥器，并允许〜15-20最小解冻。离开试样干燥器中不再将干燥样品，，并降低DESI效率。一旦解冻，但是，在时间的关系，DESI离子源的优化工作将与组织样本，将样品解冻的期间作为一个理想的时间用于该设备的初始化。 <ol><li>使用作为DESI溶剂与1％乙酸的乙腈，打开取5μl/分钟的流速注射泵。较低的流速将降低的的DESI喷有效像素尺寸的影响点侧的，但更高的流速可能是必要的足够的灵敏度。因此，流量（通常1-5微升/分钟）应该为每个应用程序和所需的灵敏度和分辨率进行了优化。确保注射器在给定的流量的全面优化和成像包含足够的溶剂。 </li><li>一旦溶剂源尖端出现滴水，打开氮气雾化气体与PRESSURE 160磅。 </li><li>打开高压电源连接到离子源申请3,600 V。 </li></ol></li><li>装载的运动舞台上的幻灯片，并开始离子源和MS优化</li></ol> 2。 DESI优化<ol><li> DESI系统包括源探针（参见图2），注射器，泵或替代溶剂输送系统，压缩氮气，翻译样品台和安装。继续进行优化DESI源质谱仪​​一次已经调整为适当的质量范围和分析物。 <ol><li>如果源组件尚未尚未打开，请参阅第1.5.1-3的方向。 </li><li>这些部分中的所讨论的变量被推荐为生物组织成像。然而，这些变量进行优化，以不同类型的组织或样本。已报告的想象中的替代溶剂，流速，雾化气体的压力和电压的生物组织16-18纳克</li></ol></li><li>确保初期DESI源不接触载玻片上，而不是冲着任何部分组织部分。这将确保探头的初始优化过程中不被损坏，不会污染任何接触到的组织样本集的一部分。 <ol><li>一个很好的起点位置是从样品表面的前端3毫米到5毫米，从MS毛细管进样口，其中探头的角度55°相对于样品表面。内毛细血管的DESI探头应延长〜1毫米超越外毛细管所有这些参数将被优化</li></ol></li><li> MS接口毛细管应该有一个收集〜15°的角度相对于最佳的离子转移到样品表面。调整阶段高度，从而MS对样品表面的毛细管悬停在毛细管应该是小于1 mm的表面，尽量靠近不接触。 </li><li>对齐的DESI探头在x维度相对于向MS毛细管进样口，使它们直接符合。 </li><li>调整的y和z的的DESI源的最佳灵敏度，使用额外的组织切片的位置。注意DESI探针是针对一个样本区域，它将最终解吸和电离当这发生时，在这方面，信号的分析物将逐渐减小。重要的是尽可能快地执行此步骤。 <ol><li>因此，整个优化过程中，需要移动的喷射头的下面，以确保新鲜样品进行分析，然后在信号中的任何变化，由于源优化，而不是样品耗​​尽样品。然而，考虑到不同区域的组织的各种化学组合物的离子强度在整个组织切片，直接的比较，虽然不完全准确。丘脑的大脑提供一个相当大的大小的区域的更为一致的化学成分，但仍然没有完全统一。或者，标记红色的骗子，其中包含的染料若丹明6G（[M] +，米/ Z 443），在载玻片上相差的样品提供了一个强有力的MS响应由DESI也可以用于优化，但给出了不同的样品形式和分析物，这些条件可能仍然没有建立一个理想的生物组织相关。 </li><li>推荐Ÿ源尖和毛细管入口之间的分离:〜4毫米，推荐Ž分离针尖和样品表面:〜1.5毫米之间。虽然每个实验设置这些参数会略有不同。 </li><li>考虑到探头的几何形状和角度，以考虑到，y和z维度的位置，相对于溶剂和含被分析物的液滴的有效传输是相互关联的。在一个变量的改变将需要在后续的修改，以保持相同的喷流冲击位置和其传动角。 </li></ol></li><li>距离调整为从外最大的灵敏度，影响最小的光点尺寸的信号源毛细管内的毛细管挤出。注意:确保高电压是关闭，而这种调整。 </li><li>时观察到的最大信号源几何体将被认为是最优的。 <ol><li>第2.5-2.6优化的变量是相互关联的，因此，调整一个固有的需要进行重新调整，以维持必要的源样品入口的几何形状，或调整第1.5.1节中所讨论的条件3。 </li></ol></li><li>此外，加热元件周围的传输毛细管到质谱仪促进在电离过程中产生的带电分析物的液滴的去溶剂化，可以提高灵敏度。在这里，我们用绳子加热器盘绕在转让毛细管设置为100℃。 </li></ol> 3。布甲É成像<ol><li>在成像过程中，在样品台上将样品下方的的DESI探针和质谱入口毛细管和所有其他组件保持静止。舞台上的运动参数，应选择基础上的DESI影响羽规模和最佳的灵敏度。 <ol><li>较大的影响羽流将导致较高的离子强度，因为更多的样品被解吸，但是成像，它也将在更大范围内的像素尺寸和图像分辨率差从而导致。 </li></ol></li><li>本实验中使用的翻译阶段是建立和控制由LabView的程序，允许控制速度和给定尺寸的图像的行间距的光栅。本实验中使用的载物台的运动控制VI提供omnispect.bme.gatech.edu。另外，市售的的DESI源和阶段可以用于如2D自动化全方位喷雾源从Prosolia公司（印第安纳波利斯，IN USA）。 <ol><li>对于脑组织一典型的图像大小为10 x 15毫米，并使用阶段的运动参数，图像将需要约3小时。 <ol><li>编程LabView的VI或等效控制软件的期望的成像条件下，使用一个阶段的扫描速度为80微米/秒，和行线之间的间距为200μm。当使用的全喷雾源，运动参数应设置为100-200微米/秒的扫描速度和线间距为200μm。请注意，根据所希望的像素数在每个维度和必要的灵敏度，这些运动参数是可以改变的。 <ol><li>一个较小的线间距已被证明能提高图像质量，与扫描速度具有以下的效果。25，但要注意，这并不一定表明，提高了图像的分辨率，它是显着的，因为这是强烈地依赖于影响光斑大小。较慢的扫描速度和更小的行距将显着增加分析时间，因此必须优化作业捷思为每个不同的组织或样本。 </li></ol></li><li>与MS采集光谱扫描/秒，并作为1个像素/扫描产生的图象设定为1，阶段速度定义在x维度上的像素大小，行间距，而决定的像素大小中的y维度。虽然真正的像素的图像尺寸大于这是由于喷雾传播，速度较慢的运动允许更多的时间进行解吸和分析物的检测。 </li></ol></li><li>给目录期间要记录在LabVIEW VI图像的位置和时间的文件的路径和文件名。 </li></ol></li><li>在制备用于质谱数据采集，计算成像所需的总时间。本集团所用的Labview程序自动提供此值，但如果使用替代阶段控制，以此计算的MS数据采集的设置是必要的。 <ol><li>当使用全向自动喷雾源和阶段中，每行的图像获取在灵敏度独立运行。时间每行所需的给定的成像尺寸的行数指的是使用全喷雾要输入到质谱仪的软件的控制软件。 </li></ol></li><li>确保质谱仪的扫描速度为1频谱/秒，和设置在仪器的采集时间，以计算出的总时间相匹配。 <ol><li>设置光谱扫描速度为1频谱/秒。 </li><li>简介模式采集数据，并确保在m / z范围是适当的利益的物种。记住DESI也产生多电荷分析，在ESI。 </li><li>设置采集时间，以配合由LabVIEW图像总时间。 </li></ol></li><li>喷流冲击点在成像区域左上角的位置。 </li><li>同时开始的MS数据采集和舞台运动。 </li><li>数据采集​​结束后，返回到待机模式质谱仪。 </li><li>关闭高电压DESI源。 </li><li>转ØFF氮气。 </li><li>关闭注射泵。 </li></ol> 4。图像处理<ol><li>质谱数据，必须在结合阶段的时间和位置数据保存为一个文本文件通过LabView的"折叠"成2D图像相关联的坐标的像素与相应的光谱。这里呈现的图像处理采用基于Matlab软件是免费提供:omnispect.bme.gatech.edu。如果数据采集采用全方位喷雾源，萤火虫数据的转换软件是用来创建多维数据集的图像可视化在BioMAP（www.maldi msi.org）。 </li><li>所记录的色谱对于数据上JEOL AccuTOF。质谱仪收购，必须出口。CDF格式进行进一步的分析。 <ol><li>使用DataManager的内MassCenter，首先将采集的数据到centroided数据（工具→习得数据转换重心）。然后将数据导出。CDF格式通过使用键盘组合键Ctrl + Shift + E键FOL然后再由"工具"→"导出。 </li></ol></li><li>上传原料CDF质谱数据和两个文本文件，位置和时间，的OmniSpect网站。萤火虫处理的图像数据可以可视化BioMAP。 <ol><li>可以直接在网站上进行进一步处理的数据包括非负矩阵分解的统计分析或绘制一个单一离子的利益。 </li><li>另外，原始数据立方体可以导出在Matlab分析。 </li></ol></li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thin-Layer+Chromatography+and+Mass+Spectrometry+Coupled+Using+Desorption+Electrospray+Ionization.">Thin-Layer Chromatography and Mass Spectrometry Coupled Using Desorption Electrospray Ionization.</a> Anal. Chem. 77, 1207-1215 (2005).</li><li>Lane, A. L., 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=Desorption+electrospray+ionization+mass+spectrometry+reveals+surface-mediated+antifungal+chemical+defense+of+a+tropical+seaweed.">Desorption electrospray ionization mass spectrometry reveals surface-mediated antifungal chemical defense of a tropical seaweed.</a> Proc. Natl. Acad. Sci. 106, 7314-7319 (2009).</li><li>Kertesz, V., Van Berkel, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+and+Surface+Alignment+Considerations+in+Chemical+Imaging+with+Desorption+Electrospray+Mass+Spectrometry.">Scanning and Surface Alignment Considerations in Chemical Imaging with Desorption Electrospray Mass Spectrometry.</a> Anal. Chem. 80, 1027-1032 (2008).</li><li>Kertesz, V., Van Berkel, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+imaging+resolution+in+desorption+electrospray+ionization+mass+spectrometry.">Improved imaging resolution in desorption electrospray ionization mass spectrometry.</a> Rapid Commun. Mass Spectrom. 22, 2639-2644 (2008).</li><li>Campbell, D., Ferreira, C., Eberlin, L., Cooks, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+spatial+resolution+in+the+imaging+of+biological+tissue+using+desorption+electrospray+ionization.">Improved spatial resolution in the imaging of biological tissue using desorption electrospray ionization.</a> Anal. Bioanal. Chem. 404, 389-398 (2012).</li><li>Chaurand, P., Cornett, D. S., Angel, P. M., 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=From+Whole-body+Sections+Down+to+Cellular+Level,+Multiscale+Imaging+of+Phospholipids+by+MALDI+Mass+Spectrometry.">From Whole-body Sections Down to Cellular Level, Multiscale Imaging of Phospholipids by MALDI Mass Spectrometry.</a> Mol. Cell. Proteomics. 10, (2011).</li><li>Dill, A., Eberlin, L., Costa, A., Ifa, D., Cooks, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+quality+in+tissue+analysis+using+desorption+electrospray+ionization.">Data quality in tissue analysis using desorption electrospray ionization.</a> Anal. Bioanal. Chem. 401, 1949-1961 (2011).</li><li>Jackson, S. N., Wang, H. -. Y. J., Woods, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Profiling+of+Lipid+Distribution+in+Brain+Tissue+Using+MALDI-TOFMS.">Direct Profiling of Lipid Distribution in Brain Tissue Using MALDI-TOFMS.</a> Anal. Chem. 77, 4523-4527 (2005).</li><li>Jackson, S. N., 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=MALDI-ion+mobility-TOFMS+imaging+of+lipids+in+rat+brain+tissue.">MALDI-ion mobility-TOFMS imaging of lipids in rat brain tissue.</a> J. Mass Spectrom. 42, 1093-1098 (2007).</li><li>Wang, H. -. Y. J., Post, S. N. J. J., Woods, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+minimalist+approach+to+MALDI+imaging+of+glycerophospholipids+and+sphingolipids+in+rat+brain+sections.">A minimalist approach to MALDI imaging of glycerophospholipids and sphingolipids in rat brain sections.</a> Int. J. Mass Spectrom. 278, 143-149 (2008).</li><li>Wu, B., Becker, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+elements+and+molecules+in+biological+tissues+and+cells+in+the+low-micrometer+and+nanometer+range.">Imaging of elements and molecules in biological tissues and cells in the low-micrometer and nanometer range.</a> Int. J. Mass Spectrom. 307, 112-122 (2011).</li><li>Eberlin, L. S., Ifa, D. R., Wu, C., Cooks, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Vizualization+of+Mouse+Brain+by+Lipid+Analysis+Using+Ambient+Ionization+Mass+Spectrometry.">Three-Dimensional Vizualization of Mouse Brain by Lipid Analysis Using Ambient Ionization Mass Spectrometry.</a> Angew. Chem. Int. Ed. 49, 873-876 (2010).</li><li>Seeley, E. H., 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=3D+Imaging+by+Mass+Spectrometry:+A+New+Frontier.">3D Imaging by Mass Spectrometry: A New Frontier.</a> Anal. Chem. 84, 2105-2110 (2012).</li><li>Nemes, P., Barton, A. A., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Imaging+of+Metabolites+in+Tissues+under+Ambient+Conditions+by+Laser+Ablation+Electrospray+Ionization+Mass+Spectrometry.">Three-Dimensional Imaging of Metabolites in Tissues under Ambient Conditions by Laser Ablation Electrospray Ionization Mass Spectrometry.</a> Anal. Chem. 81, 6668-6675 (2009).</li><li>Pulfer, M., Murphy, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospray+mass+spectrometry+of+phospholipids.">Electrospray mass spectrometry of phospholipids.</a> Mass Spectrom. Rev. 22, 332-364 (2003).</li><li>Han, X., Holtzman, D. M., McKeel, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmalogen+deficiency+in+early+Alzheimer's+disease+subjects+and+in+animal+models:+molecular+characterization+using+electrospray+ionization+mass+spectrometry.">Plasmalogen deficiency in early Alzheimer's disease subjects and in animal models: molecular characterization using electrospray ionization mass spectrometry.</a> J. Neurochem. 77, 1168-1180 (2001).</li><li>Murphy, E. J., Schapiro, M. B., Rapoport, S. I., Shetty, H. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+composition+and+levels+are+altered+in+down+syndrome+brain.">Phospholipid composition and levels are altered in down syndrome brain.</a> Brain Res. 867, 9-18 (2000).</li><li>Han, X., 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=Alterations+in+Myocardial+Cardiolipin+Content+and+Composition+Occur+at+the+Very+Earliest+Stages+of+Diabetes:+A+Shotgun+Lipidomics+Study.">Alterations in Myocardial Cardiolipin Content and Composition Occur at the Very Earliest Stages of Diabetes: A Shotgun Lipidomics Study.</a> Biochemistry. 46, 6417-6428 (2007).</li></ol>

作者宣称，他们有没有竞争经济利益。

DESI源的几何形状的优化MSI实验成功的关键。促进的系统的对应的多个变量直接影响灵敏度和图像分辨率。如果在优化过程中，实验者获得信号有困难，我们建议使用Sharpie笔现货为基准，在幻灯片上绘制红色染料罗丹明6G，M / Z 443，在正离子模式，并产生一个强烈的信号，可用于初始优化。为DESI此外，溶剂的选择是至关重要的灵敏度，分析物传输和电离取决于从表面到所形成的薄膜的被分析物的?...

A correction was made to <a href="http://www.jove.com/video/50575" target="_parent">Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry</a>. There was an error with an author's name. The author's surname was appended with a missing character:
Rachel V. Bennett
instead of:
Rachel V. Bennet

Erratum: Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

无针对性的质谱成像，有利于发现和假说产生应用化学信息的收购。目标成像，在另一方面，还公知的化学，可以方便的灵敏度和选择性的增加，通过特定的方法的发展。质谱成像（MSI）是最常见的组织上进行二次离子质谱（SIMS），使用MALDI 2和环境离子化技术，包括解吸电喷雾电离（DESI），激光烧蚀电喷雾离子化（LAESI），4， 5和液体微结表面取样探头（LMJ-SSP）6 MALDI和SIMS，样品标本进行物理删除，并有平而薄，因为他们是在高真空下进行分析。 MALDI需要涂层的辐射吸收矩阵的样品，样品的制备增加一个额外的，繁琐的步骤。 SIMS具有最高的横向分辨率，但高度高能粒子轰击导致广泛的分子碎片。因此，微星环境的方法填补一个利基，用最少的样品制备软分析是可取的。然而，迄今为止，所有的方法仍然由平坦的样品表面的要求的限制。 DESI 7采用了气动辅助充电冲着样品表面解吸和电离分析物的溶剂喷雾解吸电离和后续DESI工作模型被称为"液滴回升模型"8-10主要液滴带电DESI探头所产生的碰撞的表面润湿，并形成薄膜，到其中的分析物被溶解的固-液微萃取机制8随后的液滴碰撞的结果中包含从表面提取的物质的二次液滴动量转移和起飞9,10，气体最终相离子被认为是离子蒸发，电荷残基模型或其他模型，11后通过ESI-状突起DESI然而，精确的离子形成过程中仍有待实验证明。的溶解度12的 DESI灵敏度是强烈地依赖于中的分析物的喷雾溶剂，解吸依赖于本地化的微萃取13 一起使用时，一个由软件控制的样品台，样品与车道步进DESI电离探针下方单向扫描，通过时域的m / z的信息是相关的化学物种的空间分布（ 图1）。 DESI-MSI原理实验报告由Van Berkel的凯尔泰斯在2006年以来的首次证明，14的技术已经相当成熟，15血脂分析，药物代谢产物3,16，17,18 disea报告应用硒的生物标志物，脑组织，19 3,18,20肺组织，肾组织18 18睾丸组织，18个肾上腺，17薄层层析板，21和藻类表面22例行DESI-MSI所获得的图像分辨率100-200微米，最终由喷雾提取的有效表面积，但已报告低至40微米的分辨率。23-25 ​​日这样的分辨率和便于分析，使DESI-MSI适合的快速和简单的分析生物组织样本的表面区域在0.5-5厘米2的范围内，使宝贵的空间信息的收购，以更好地理解生物过程26。在这里，DESI-MSI一个典型的应用程序作为一个例子，我们进行了成功的实验，涉及成像大鼠脑组织中脂质的程序细节。在协议中两个最重要的步骤是组织制备27和DESI离子源优化，如下所述。

<table border="1" fo:keep-together.within-page="always">
		<tbody>
		 <tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Tissue-Tek O.C.T. Compound</td>
			<td>Sakura-Finetek</td>
			<td>4583</td>
			<td><a href="http://www.sakuraeu.com/products/showitem.asp?cat=11&amp;subcat=48" target="_blank">http://www.sakuraeu.com/products/showitem.asp?cat=11&amp;subcat=48</a></td>
		 </tr>
		 <tr>
			<td>Acetonitrile</td>
			<td>EMD</td>
			<td>AX0156-6</td>
			<td>OmniSolv, LC-MS Grade</td>
		 </tr>
		 <tr>
			<td>Acetic Acid</td>
			<td>Sigma Aldrich</td>
			<td>695092-500 ml</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Cryostat microtome</td>
			<td>Thermo Scientific</td>
			<td>CryoStar* NX70</td>
			<td>Any available microtome can be used for tissue sectioning <a href=" http://www.thermoscientific.com/ecomm/servlet/productsdetail?productId=13958375&amp;groupType=PRODUCT&amp;searchType=0&amp;storeId=11152&amp;from=search&amp;ca=cryostar" target="_blank"> http://www.thermoscientific.com/ecomm/servlet/productsdetail?productId=13958375&amp;groupType=PRODUCT&amp;searchType=0&amp;storeId=11152&amp;from=search&amp;ca=cryostar</a></td>
		 </tr>
		 <tr>
			<td>Omni Spray;DESI Spray Head</td>
			<td>Prosolia Inc.</td>
			<td></td>
			<td>Can also use the 2-D Omni Spray; Source kit instead of assembling components of imaging experiment <a href=" http://www.prosolia.com/sources.php" target="_blank"> http://www.prosolia.com/sources.php</a></td>
		 </tr>
		 <tr>
			<td>High Voltage Power Supply</td>
			<td>Stanford Research Systems, Inc.</td>
			<td>PS350/5000V-25W</td>
			<td><a href="http://www.thinksrs.com/products/PS300.htm" target="_blank">http://www.thinksrs.com/products/PS300.htm</a></td>
		 </tr>
		 <tr>
			<td>Rope heater, RTD, controller</td>
			<td>Omega</td>
			<td></td>
			<td><a href="http://www.omega.com/toc_asp/subsectionSC.asp?subsection=M02&amp;book=Heaters" target="_blank">http://www.omega.com/toc_asp/subsectionSC.asp?subsection=M02&amp;book=Heaters</a></td>
		 </tr>
		 <tr>
			<td>Labview</td>
			<td>National Instruments</td>
			<td>Version 7.1 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Translational stage</td>
			<td>Prior Scientific</td>
			<td>Optiscan II</td>
			<td><a href="http://www.prior.com/productinfo_auto_motorized_optiscan.html" target="_blank">http://www.prior.com/productinfo_auto_motorized_optiscan.html</a></td>
		 </tr>
		 <tr>
			<td>AccuTOF Mass Spectrometer</td>
			<td>JEOL</td>
			<td>JMS-T100LC</td>
			<td>Can use any mass spectrometer equipped with an extended capillary atmospheric pressure interface</td>
		 </tr>
		</tbody>
 </table>

imaging of biological tissues by desorption electrospray ionization mass spectrometry

质谱成像（MSI）提供不相关的分子调查在几百到几十微米规模的生物组织具有最高特异性和空间分辨率的信息。当在环境条件下，进行样​​品的前处理变得不必要了，从而简化了协议，而得到的信息保持高品质。解吸电喷雾电离（DESI）是一个基于喷雾环境的MSI的技术，它允许在露天的表面，即使是在体内直接采样。一起使用时，一个由软件控制的样品台，样品下方光栅DESI电离探针，通过时域的m / z的信息是相关的化学物种的空间分布。的DESI-MSI输出的保真度取决于源的方向和定位，对于样品表面和质谱仪入口。在此，我们回顾一下如何准备组织切片DESI我的maging和额外的实验条件下，直接影响图像质量。具体来说，我们描述成像大鼠脑组织切片的DESI-MSI的协议。

图3示出了代表性的频谱得到的未经处理的大鼠的脑切片。质谱在正离子模式，主要是由于其高离子化效率（归因于带正电的季铵基团）的磷脂酰胆碱。组织切片的总离子图像也显示在图3中 ，示出在整个脑切片的丰富的信号。通过文献比较表1主要血脂检测确定。 例如血脂的空间分布（ 图4）显示不同的卵磷脂物种的相对丰?...

Watch this Scientific Journal Video about Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry at JoVE.com

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

电喷雾解吸电离质谱（DESI-MS）是一个环境样本，包括生物组织，可以用最少的样品制备成像方法。通过离子探针光栅下面的示例，这个喷雾基于技术组织切片内辨别分子功能提供了足够的空间分辨率。

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 ...

imaging-biological-tissues-desorption-electrospray-ionization-mass

Research

JoVE Journal

Bioengineering

18.5K 次观看.  Georgia Institute of Technology. 电喷雾解吸电离质谱（DESI-MS）是一个环境样本，包括生物组织，可以用最少的样品制备成像方法。通过离子探针光栅下面的示例，这个喷雾基于技术组织切片内辨别分子功能提供了足够的空间分辨率。

视频: Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

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

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

高分辨率三维成像 EX - VIVO生物样品显微CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

原子力显微镜成像的细菌固定化

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

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 ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

由多光谱成像流式细胞仪分析细胞内化纳米粒子和细菌

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

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 ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

从藻类水热液化使用二维气相色谱时间飞行质谱法水提取部分的定性鉴定

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

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

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such methods, where features of matrix-assisted laser desorption/ionization (MALDI) (e.g., pulsed nature of desorption) and electrospray ionization (ESI) (e.g., soft-ionization) are combined. One of the major advantages of MALDESI is its inherent versatility. In MALDESI experiments, an ultraviolet (UV) or infrared (IR) laser can be used to resonantly excite an endogenous or exogenous matrix. The choice of matrix is not analyte dependent, and depends solely on the laser wavelength used for excitation. In IR-MALDESI experiments, a thin layer of ice is deposited on the sample surface as an energy-absorbing matrix. The IR-MALDESI source geometry has been optimized using statistical design of experiments (DOE) for analysis of liquid samples as well as biological tissue specimens. Furthermore, a robust IR-MALDESI imaging source has been developed, where a tunable mid-IR laser is synchronized with a computer controlled XY translational stage and a high resolving power mass spectrometer. A custom graphical user interface (GUI) allows user selection of the repetition rate of the laser, number of shots per voxel, step-size of the sample stage, and the delay between the desorption and scan events for the source. IR-MALDESI has been used in variety of applications such as forensic analysis of fibers and dyes and MSI of biological tissue sections. Distribution of different analytes ranging from endogenous metabolites to exogenous xenobiotics within tissue sections can be measured and quantified using this technique. The protocol presented in this manuscript describes major steps necessary for IR-MALDESI MSI of whole-body tissue sections.

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 ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

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

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

工程三维上皮组织细胞外基质中嵌入

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

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).