Entwicklung und Anwendung laseroptischer Methoden zum Screening von Membranen lebender Zellen

A setup for fluorescence measurements of surfaces of biological samples, in particular the plasma membrane of living cells, is described. The method is based on splitting of a laser beam and multiple total internal reflections (TIR) within the bottom of a microtiter plate, such that up to 96 individual samples are illuminated simultaneously by evanescent electromagnetic fields. The presented microplate-TIRF-reader combines two different screening procedures related to high throughput screening (HTS) and high content screening (HCS). HTS allows a rapid measurement of large sample numbers based on fluorescence intensity, whereas by HCS a high content of information from a single sample based on the parameters fluorescence lifetime (fluorescence lifetime screening) and fluorescence anisotropy (fluorescence lifetime polarization screening) is achieved. Both screening systems were validated using cultivated cells incubated with different fluorescent markers (e.g. NBD-cholesterol, rhodamine 123) as well as stably transfected cells expressing a fluorescent membrane-associating protein (e.g. EYFP-mem, ECFP-mem). In addition, particularly with regard of potential pharmaceutical applications, the kinetics of the intracellular translocation of a fluorescent protein kinase c fusion protein upon stimulation of the cells was determined. Further, a caspase sensor based on Förster Resonance Energy Transfer (FRET) between fluorescent proteins was tested. Enhanced cyan fluorescent protein (ECFP) anchored to the inner leaflet of the plasma membrane of living cells transfers its excitation energy via a spacer (DEVD) to an enhanced yellow fluorescent protein (EYFP). Upon apoptosis DEVD is cleaved by caspase-3, and energy transfer is disrupted, as proven by changes in fluorescence intensity and decay times.