Cell-substrate contacts illuminated by total internal reflection fluorescence.

Abstract

A technique for exciting fluorescence exclusively from regions of contact between cultured cells and the substrate is presented. The technique uses the evanescent wave of a totally internally reflecting laser beam to excite only those fluorescent molecules within 1 light wavelength or less of the substrate surface. Demonstrations of this technique are given for 2 types of cell cultures: rat primary myotubes with acetylcholine receptors labeled by fluorescent .alpha.-bungarotoxin and human skin fibroblasts labeled by a fluorescent lipid probe. Total internal reflection fluorescence examination of cells appear to have promising applications, including visualization of the membrane and underlying cytoplasmic structures at cell-substrate contacts, dramatic reduction of autofluorescence from debris and thick cells, mapping of membrane topography, and visualization of reversibly bound fluorescent ligands at membrane receptors.