LASER-ASSISTED PATCH CLAMPING - A METHODOLOGY

Laser microsurgery can be used to perform both cell biological manipul ations, such as targeted cell ablation, and molecular genetic manipula tions, such as genetic transformation and chromosome dissection. In th is report, we describe a laser microsurgical method that can be used e ither to ablate single cells or to ablate a small area (1-3 mu m diame ter) of the extracellular matrix. In plants and microorganisms, the ex tracellular matrix consists of the cell wall. While conventional patch clamping of these cells, as well as of many animal cells, requires en zymatic digestion of the extracellular matrix, we illustrate that lase r 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 cons truction and use of an economical laser microsurgery system, including procedures for single cell and targeted cell wall ablation. This meth odology 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.