Laser capture microdissection of single cells from complex tissues

Laser capture microdissection (LCM) is a nerv method used to select and pro cure cell clusters from tissue sections. Once captured, the DNA, RNA or pro tein can be easily extracted from the isolated cells and analyzed by conven tional PCR, reverse transcription (RT)-PCR or polyacrylamide gel electropho resis, including protein zymography far specific macromolecular changes. In LCM, a thermoplastic polymer coating [ethylene vinyl acetate (EVA)] attach ed to a rigid support is placed in contact with a tissue section. The EVA p olymer over microscopically selected cell clusters is precisely activated b y 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 ce ll 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 macromolec ular analysis in many laboratories around the world. However reliable and p recise capture of individual cells fron tissue sections has been difficult to perform, with the current LCM instruments. In this report, we describe t he capture of individual cells with a new NIH LCM microscope, which epi-irr adiates the EVA polymer overlying individual cells with 1-ms laser pulses f ocused to 6 mu m. A computer-controlled aml precisely positions a 40-mu m-w ide strip of a cylindrical EVA surface onto a sample with a light contact f orce (ca. 0.1 g). The small contact force and contact area on the film on t he sample diminishes nonspecific transfer to negligible levels. By slightly rotating the cylinder to provide a renewable transfer surface, concentrati on of 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.