Nahfeldoptische Untersuchungen an biologischen Systemen im ultravioletten und sichtbaren Wellenlängenbereich

____________________________________________________________________________________________________ Abstract The aim of the present work was to demonstrate the capability and feasibility of scanning near-field optical microscopy (SNOM) to study the penetration of UV-light absorbing or fluorescent molecules into human skin and hair fibres. For this purpose a commercial SNOM was modified, in order to operate in the UV wavelength range in transmission mode. In addition, the system was expanded for a simultaneous detection of fluorescence and transmission signals. UV-absorbers were imaged and localized on glass slides due to their specific absorption at 300nm or 350nm. For the penetration study human skin was chemically fixed, dehydrated in ethanol, and embedded in epon resin. 200nm thin sections of treated and untreated skin were investigated by SNOM. In such thin sections UVabsorber molecules could not be unequivocally identified and localized because of strong variations in the transmission signal. The signal variations are caused by other effects such as thickness variations, scattering, and surface profile dendent influences in SNOM transmission mode. Immuno labeled thin sections of human skin against cytokeratin10 investgated by SNOM in fluorescence mode revealed distinct and very sharp images of the cytokeratin10 distribution in human epidermis. On the one side this allows to localize proteins and lipids more precisely than confocal laser scanning microscopy (CLSM) imaging and on the other side this technique offers an additional way to investigate penetrating molecules if antibodies exist to the substance of interest. The high potential of SNOM was also demonstrated on thin sections of fluorophor stained human hair fibres. The results showed the distribution of the dye inside the hair with a resolution of about 130nm. This resolution is higher than classical light microscopy can ever achieve. SNOM images in fluorescence mode showed identical contrast and signal variations as in CLSM and hence no topographical nor scattering effects seems to disturbe the fluorescence signal. Therefore, SNOM is an alternative to classical methods for a penetration study of fluorophors or fluorescent labeled substances. A great advantage of SNOM imaging is the possibility to image simultaneously topography, transmission, and fluorescence of the same sample area, whereas to generate these various informations from one specimen with other techniques different microscopes such as electronmicroscopes, atomic force microscopes, or confocal laser scanning microscopes are necessary.The aim of the present work was to demonstrate the capability and feasibility of scanning near-field optical microscopy (SNOM) to study the penetration of UV-light absorbing or fluorescent molecules into human skin and hair fibres. For this purpose a commercial SNOM was modified, in order to operate in the UV wavelength range in transmission mode. In addition, the system was expanded for a simultaneous detection of fluorescence and transmission signals. UV-absorbers were imaged and localized on glass slides due to their specific absorption at 300nm or 350nm. For the penetration study human skin was chemically fixed, dehydrated in ethanol, and embedded in epon resin. 200nm thin sections of treated and untreated skin were investigated by SNOM. In such thin sections UVabsorber molecules could not be unequivocally identified and localized because of strong variations in the transmission signal. The signal variations are caused by other effects such as thickness variations, scattering, and surface profile dendent influences in SNOM transmission mode. Immuno labeled thin sections of human skin against cytokeratin10 investgated by SNOM in fluorescence mode revealed distinct and very sharp images of the cytokeratin10 distribution in human epidermis. On the one side this allows to localize proteins and lipids more precisely than confocal laser scanning microscopy (CLSM) imaging and on the other side this technique offers an additional way to investigate penetrating molecules if antibodies exist to the substance of interest. The high potential of SNOM was also demonstrated on thin sections of fluorophor stained human hair fibres. The results showed the distribution of the dye inside the hair with a resolution of about 130nm. This resolution is higher than classical light microscopy can ever achieve. SNOM images in fluorescence mode showed identical contrast and signal variations as in CLSM and hence no topographical nor scattering effects seems to disturbe the fluorescence signal. Therefore, SNOM is an alternative to classical methods for a penetration study of fluorophors or fluorescent labeled substances. A great advantage of SNOM imaging is the possibility to image simultaneously topography, transmission, and fluorescence of the same sample area, whereas to generate these various informations from one specimen with other techniques different microscopes such as electronmicroscopes, atomic force microscopes, or confocal laser scanning microscopes are necessary. Inhaltsverzeichnis _____________________________________________________________________________________________________