Golubeva Yelena, Salcedo Rosalba, Mueller Claudius, Liotta Lance A, Espina Virginia
National Cancer Institute-Frederick/SAIC, Frederick, MD, USA.
Methods Mol Biol. 2013;931:213-57. doi: 10.1007/978-1-62703-056-4_12.
Laser capture microdissection (LCM) allows the precise procurement of enriched cell populations from a heterogeneous tissue, or live cell culture, under direct microscopic visualization. Histologically enriched cell populations can be procured by harvesting cells of interest directly or isolating specific cells by ablating unwanted cells. The basic components of laser microdissection technology are (a) visualization of cells via light microscopy, (b) transfer of laser energy to a thermolabile polymer with either the formation of a polymer-cell composite (capture method) or transfer of laser energy via an ultraviolet laser to photovolatize a region of tissue (cutting method), and (c) removal of cells of interest from the heterogeneous tissue section. The capture and cutting methods (instruments) for laser microdissection differ in the manner by which cells of interest are removed from the heterogeneous sample. Laser energy in the capture method is infrared (810 nm), while in the cutting mode the laser is ultraviolet (355 nm). Infrared lasers melt a thermolabile polymer that adheres to the cells of interest, whereas ultraviolet lasers ablate cells for either removal of unwanted cells or excision of a defined area of cells. LCM technology is applicable to an array of applications including mass spectrometry, DNA genotyping and loss-of-heterozygosity analysis, RNA transcript profiling, cDNA library generation, proteomics discovery, and signal kinase pathway profiling. This chapter describes LCM using an Arcturus(XT) instrument for downstream protein sample analysis and using an mmi CellCut Plus® instrument for RNA analysis via NanoString technology.
激光捕获显微切割（LCM）技术能够在直接显微镜观察下，从异质性组织或活细胞培养物中精确获取富集的细胞群体。通过直接采集感兴趣的细胞或通过去除不需要的细胞来分离特定细胞，可以获得组织学上富集的细胞群体。激光显微切割技术的基本组成部分包括：（a）通过光学显微镜观察细胞；（b）将激光能量传递给热不稳定聚合物，形成聚合物 - 细胞复合物（捕获法），或者通过紫外激光传递激光能量使组织区域光挥发（切割法）；（c）从异质性组织切片中去除感兴趣的细胞。激光显微切割的捕获和切割方法（仪器）在从异质性样品中去除感兴趣细胞的方式上有所不同。捕获法中的激光能量为红外光（810 nm），而切割模式下的激光为紫外光（355 nm）。红外激光使热不稳定聚合物熔化，该聚合物会附着在感兴趣的细胞上，而紫外激光则通过烧蚀细胞来去除不需要的细胞或切除特定区域的细胞。LCM技术适用于一系列应用，包括质谱分析、DNA基因分型和杂合性缺失分析、RNA转录本分析、cDNA文库构建、蛋白质组学发现以及信号激酶途径分析。本章介绍了使用Arcturus（XT）仪器进行下游蛋白质样品分析以及使用mmi CellCut Plus®仪器通过NanoString技术进行RNA分析的LCM技术。
