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Bioingegneria

解吸电喷雾电离质谱成像的生物组织

Published: July 12, 2013
doi:
10.3791/50575
, ,
1School of Chemistry and Biochemistry,Georgia Institute of Technology

Summary

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。

Abstract

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。

Introduction

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。

Protocol

1。组织切片商店闪光灯冷冻，在-80℃冷冻，直到整个组织准备切片。 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℃。 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）。 应用〜0.5毫升最佳切削温度化?…

Representative Results

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?…

Discussion

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?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

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。

Materials

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

Mass Spectrometry ImagingDESIDesorption Electrospray IonizationAmbient IonizationRat BrainSample PreparationSpatial ResolutionMolecular Information
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Citazione di questo articolo
Bennett, R. V., Gamage, C. M., Fernández, F. M. Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry. J. Vis. Exp. (77), e50575, doi:10.3791/50575 (2013).
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