Inaugural Dissertation - Ruprecht-Karls-Universität Heidelberg

Abstract
Photo-optical nanoscopy of biological structures with focussed,
structured and homogeneous illumination
Recently different methods of laser optical far field microscopy have been developed, which make it
possible to analyze biological structures beyond the classical limit of optical resolution. In this thesis
the three physically different approaches 4Pi-microscopy, ”
Spatially Modulated Illumination“(SMI)-
microscopy and localization microscopy have been used. The imaging properties of the 4Pi-microscope
were characterized on fluorescent nanospheres and biological samples. With SMI-microscopy the extension
of clusters of receptor proteins in Escherichia coli bacteria could be examined in direction of
the optical axis. Intrinsic properties of the fluorophores could be used in localization microscopy with
wavelength of the excitation light of 488 or 647nm to separate their signal in time and to determine their
lateral position. With this method statistical distance analysis of different receptor proteins in Escherichia
coli bacteria in comparison with simulated random distributions have been performed. The smallest
resolved inter molecule distances measured were in the range of few tens of nanometer. The standard
deviations of the mean distances were in the range of single-digit nanometers. At the motor protein complex
of the flagella of Escherichia coli the size of two ring structures (ca. 25 and 40nm in diameter) were
statistically examined. The influence of localization accuracy and detection efficiency were analyzed by
simulations of the motor protein distribution. Membrane analysis of Toxoplasma gondii Tachyzoiten by
image processing of localization microscopy data lead to an estimation for the distance between the inner
membrane complex and the outer membrane in these parasites in good agreement with results of electron
microscopy. By analysis of the distribution of the histone H3 tri-methylation at lysine 9 in embryonal mice
cells comparative studies between a wild type cell line and a knockout cell line of the Polycomb-gene
EED were realized.
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