Henriksen G H, Assmann S M
Department of Biology, Pennsylvania State University, University Park 16802, USA.
Pflugers Arch. 1997 Apr;433(6):832-41. doi: 10.1007/s004240050352.
Laser microsurgery can be used to perform both cell biological manipulations, such as targeted cell ablation, and molecular genetic manipulations, such as genetic transformation and chromosome dissection. In this report, we describe a laser microsurgical method that can be used either to ablate single cells or to ablate a small area (1-3 microns diameter) of the extracellular matrix. In plants and microorganisms, the extracellular matrix consists of the cell wall. While conventional patch clamping of these cells, as well as of many animal cells, requires enzymatic digestion of the extracellular matrix, we illustrate that laser microsurgery of a portion of the wall enables patch clamp access to the plasma membrane of higher plant cells remaining situated in their tissue environment. What follows is a detailed description of the construction and use of an economical laser microsurgery system, including procedures for single cell and targeted cell wall ablation. This methodology will be of interest to scientists wishing to perform cellular or subcellular ablation with a high degree of accuracy, or wishing to study how the extracellular matrix affects ion channel function.
激光显微手术可用于进行细胞生物学操作，如靶向细胞消融，以及分子遗传学操作，如基因转化和染色体解剖。在本报告中，我们描述了一种激光显微手术方法，该方法可用于消融单个细胞或消融细胞外基质的小区域（直径1 - 3微米）。在植物和微生物中，细胞外基质由细胞壁组成。虽然对这些细胞以及许多动物细胞进行传统的膜片钳记录需要对细胞外基质进行酶消化，但我们证明，对细胞壁的一部分进行激光显微手术能够使膜片钳记录到仍处于其组织环境中的高等植物细胞的质膜。接下来是对一种经济的激光显微手术系统的构建和使用的详细描述，包括单细胞和靶向细胞壁消融的程序。该方法将引起希望以高精度进行细胞或亚细胞消融，或希望研究细胞外基质如何影响离子通道功能的科学家的兴趣。
