Corgiat Brian A, Mueller Claudius
Center for Applied Proteomics and Molecular Medicine, George Mason University, 10920 George Mason Circle, MS1A9, Manassas, VA, 20110, USA.
Methods Mol Biol. 2017;1606:115-132. doi: 10.1007/978-1-4939-6990-6_8.
Laser capture microdissection (LCM) is a technique that allows procurement of an enriched cell population from a heterogeneous tissue sample under direct microscopic visualization. Fundamentally, laser capture microdissection consists of three main steps: (1) visualizing the desired cell population by microscopy, (2) melting a thermolabile polymer onto the desired cell populations using infrared laser energy to form a polymer-cell composite (capture method) or photovolatizing a region of tissue using ultraviolet laser energy (cutting method), and (3) removing the desired cell population from the heterogeneous tissue. In this chapter, we discuss the infrared capture method only. LCM technology is compatible with a wide range of downstream applications such as mass spectrometry, DNA genotyping and RNA transcript profiling, cDNA library generation, proteomics discovery, and signal pathway mapping. This chapter profiles the ArcturusXT laser capture microdissection instrument, using isolation of specific cortical lamina from nonhuman primate brain regions, and sample preparation methods for downstream proteomic applications.
激光捕获显微切割（LCM）是一种能够在直接显微镜观察下从异质性组织样本中获取富集细胞群体的技术。从根本上讲，激光捕获显微切割主要包括三个主要步骤：（1）通过显微镜观察所需的细胞群体；（2）使用红外激光能量将热不稳定聚合物熔化到所需的细胞群体上，以形成聚合物 - 细胞复合物（捕获法），或使用紫外激光能量使组织区域光挥发（切割法）；（3）从异质性组织中分离出所需的细胞群体。在本章中，我们仅讨论红外捕获法。LCM技术与多种下游应用兼容，如质谱分析、DNA基因分型和RNA转录谱分析、cDNA文库构建、蛋白质组学发现以及信号通路绘图。本章介绍了ArcturusXT激光捕获显微切割仪器，包括从非人灵长类动物脑区分离特定皮质层以及用于下游蛋白质组学应用的样本制备方法。
