Lunedì 31 ottobre 2005, alle ore 11.00 in aula Bianchi, Irene Upman, University of Twente, Enschede ( The Netherlands ), affronterà l’argomento.
The development of extended microscopic techniques is directly connected with the possibility to explore biomedical questions with light optical methods. A preferred technique for a fast detection of fluorescent molecules in its natural in vivo environment is the “Total Internal Reflection Fluorescent” microscopy (TIRF-microscopy). Based on the total reflection of the excitation beam, this technique allowed it to excite the fluorescence at the boundary layer with a penetration depth of a few nanometers and a minimization of the background fluorescence. Therefore an objective adapted TIRF-microscope with a “prism filter block” was realized, which allows the detection of single images in the order of 100 milliseconds. In one application the dwell time of ATP to myosin and to actomyosin was measured; in the case of myosin the expected dwell time of one second in the maximum could be confirmed. With a further microscopic technique, the “Spatially Modulated Illumination” microscopy (SMI-microscopy), measurements of the localization and the size of molecules in the fixed state in axial direction (optical axis) are possible, which are in the one/ten nanometer order respectively. With this method size measurements of protein cluster in PtK2-cells were performed. Another microscopic application is the high resolution Near-field Optical Microscopy (NSOM) in combination with single molecule detection sensitivity. NSOM, as surface sensitive technique, provides an optical resolution of ~70 nm and reduces cytoplasm fluorescence enabling single molecule detection at the cell surface. Here NSOM combined with a confocal microscope was used to investigate the nanoscale organization and the co-localization of two different epithelial cell adhesion molecules (Ep-CAM) on the membrane of human cancer cells (MCF-7). The images show that both Ep-CAM 1 and 2 accumulate preferably at the cell-cell contact regions and also express at the cell borders. Furthermore the involvement of Ep-CAMs in lipid rafts were investigated.