A time-resolved total internal reflection aqueous fluorescence (TIRAF) microscope for the investigation of cell adhesion dynamics

To investigate dynamic processes of cell adhesion on smooth and structured surfaces we developed a time-resolved TIRAF microscope with high spatial and temporal resolution. A lateral image resolution, close to the resolution limit of 223 nm, is reached using a Variable microchannel with maximum height of 210 mu m for cell cultivation in combination with a x60 oil immersion objective of numerical aperture 1.4. Using Rhodamine Green as aqueous volume marker, no significant cell membrane damage occurs within 100 min under continuous irradiation in our experimental conditions. In time-discrete experiments with L929 cells over a period of 11 h we observed interactions of adhesion areas with the surface to which the cell adhered. Short-term measurements of a defined area of a single cell containing only a few adhesion contacts showed interactions between single adhesion clusters and also putative formation of new clusters. Stationary TIRAF experiments with cells growing on structured surfaces or between microelectrodes are possible without producing additional reflections. We conclude from our first results that time-resolved TIRAF microscopy is a powerful technique for studying the mechanism of cell motion, especially under a variety of artificial conditions used to influence cell migration.