9 October 2003 Fluorescence lifetime imaging (FLIM) of membrane markers in living cells
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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.
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Herbert Schneckenburger, Michael Wagner, Martina Kretzschmar, Wolfgang S.L. Strauss, Reinhard Sailer, "Fluorescence lifetime imaging (FLIM) of membrane markers in living cells", Proc. SPIE 5139, Confocal, Multiphoton, and Nonlinear Microscopic Imaging, (9 October 2003); doi: 10.1117/12.502245; https://doi.org/10.1117/12.502245
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