Abstract
An experimental setup for fluorescence lifetime imaging (FLIM) has been combined with total internal reflection fluorescence microscopy (TIRFM) in order to detect various membrane markers within living cells. The method is established using T47D human breast cancer cells transfected by a plasmid encoding for a membrane associated yellow fluorescent protein (EYFPmem). For further measurements the mitochondrial marker rhodamine 123 (R123) as well as the membrane marker laurdan are used. With increasing concentration R123 is accumulated outside the mitochondria, in particular within the plasma membrane, whereas mitochondrial fluorescence is quenched. Fluorescence lifetime of laurdan can be used to probe membrane dynamics, in particular the phase of membrane lipids. These lipids are in a rigid gel phase at temperatures around 24°C, whereas the gel phases and a liquid crystalline phase coexist at T ≥ 30°C. This phase pattern also depends on the age and the growth phase of the cells and may play a role in the uptake of pharmaceutical agents.
© 2003 SPIE
PDF ArticleMore Like This
Petra Weber, Michael Wagner, Wolfgang S.L. Strauss, and Herbert Schneckenburger
6633_4 European Conference on Biomedical Optics (ECBO) 2007
Michael Wagner, Petra Weber, and Herbert Schneckenburger
6633_8 European Conference on Biomedical Optics (ECBO) 2007
HB Manning, DM Owen, E Auksorius, PAA de Beule, S Oddos, CB Talbot, C Dunsby, I Munro, AI Magee, MAA Neil, and PMW French
6630_44 European Conference on Biomedical Optics (ECBO) 2007