Suarez-Quian C A, Goldstein S R, Pohida T, Smith P D, Peterson J I, Wellner E, Ghany M, Bonner R F
National Institute for Child Health and Human Development, NIH, Bethesda, MD, USA.
Biotechniques. 1999 Feb;26(2):328-35. doi: 10.2144/99262rr03.
Laser capture microdissection (LCM) is a new method used to select and procure cell clusters from tissue sections. Once captured, the DNA, RNA or protein can be easily extracted from the isolated cells and analyzed by conventional PCR, reverse transcription (RT)-PCR or polyacrylamide gel electrophoresis, including protein zymography for specific macromolecular changes. In LCM, a thermoplastic polymer coating [ethylene vinyl acetate (EVA)] attached to a rigid support is placed in contact with a tissue section. The EVA polymer over microscopically selected cell clusters is precisely activated by a near-infrared laser pulse and then bonds to the targeted area. Removal of the EVA and its support from the tissue section procures the selected cell aggregates for molecular analysis. This initial NIH LCM approach using a flat transfer EVA film has been recently commercialized and has proven to be an effective routine microdissection technique for subsequent macromolecular analysis in many laboratories around the world. However, reliable and precise capture of individual cells from tissue sections has been difficult to perform with the current LCM instruments. In this report, we describe the capture of individual cells with a new NIH LCM microscope, which epi-irradiates the EVA polymer overlying individual cells with 1-ms laser pulses focused to 6 microns. A computer-controlled arm precisely positions a 40-micron-wide strip of a cylindrical EVA surface onto a sample with a light contact force (ca. 0.1 g). The small contact force and contact area on the film on the sample diminishes nonspecific transfer to negligible levels. By slightly rotating the cylinder to provide a renewable transfer surface, concentration of a distinct cell type on a single cylinder is possible. Using this novel adaptation, we demonstrate the rapid and practical capture of single cells from different types of tissue sections, including immunostained cells.
激光捕获显微切割（LCM）是一种用于从组织切片中选择并获取细胞簇的新方法。一旦捕获，DNA、RNA或蛋白质就可以很容易地从分离出的细胞中提取出来，并通过常规PCR、逆转录（RT）-PCR或聚丙烯酰胺凝胶电泳进行分析，包括用于特定大分子变化分析的蛋白质酶谱分析。在LCM中，附着在刚性支撑物上的热塑性聚合物涂层[乙烯-醋酸乙烯酯（EVA）]与组织切片接触。通过近红外激光脉冲精确激活显微镜下选定细胞簇上方的EVA聚合物，然后将其与目标区域结合。从组织切片中移除EVA及其支撑物，即可获取选定的细胞聚集体用于分子分析。美国国立卫生研究院（NIH）最初使用平面转移EVA膜的这种LCM方法最近已商业化，并且在世界各地的许多实验室中已被证明是一种用于后续大分子分析的有效的常规显微切割技术。然而，使用当前的LCM仪器很难从组织切片中可靠且精确地捕获单个细胞。在本报告中，我们描述了使用新型NIH LCM显微镜捕获单个细胞的方法，该显微镜用聚焦至6微米的1毫秒激光脉冲对覆盖单个细胞的EVA聚合物进行落射照射。计算机控制的机械臂以轻接触力（约0.1克）将圆柱形EVA表面的一条40微米宽的条带精确地放置在样品上。样品上薄膜的小接触力和接触面积将非特异性转移降低到可忽略不计的水平。通过稍微旋转圆柱体以提供可再生的转移表面，可以在单个圆柱体上富集一种独特的细胞类型。使用这种新颖的方法，我们展示了从不同类型的组织切片（包括免疫染色细胞）中快速且实际地捕获单个细胞的过程。
