Lokalisationsmikroskopie mit mehreren Farben und ihre Anwendung in biologischen Präparaten

Fluorescence microscopy is an important method for studying biological structures. By intrinsic properties of the fluorophores it is possible to separate and individually localize their signals. These quantitative position information enable an improved structural resolution and a statistic analysis of the data. In this thesis, localization microscopy was applied for multiple spectral markers within the observed biological samples. Several algorithms to compensate mechanical and chromatic shift and to correct and analyze the position data were developed. For example, the relative distribution of a structural protein of the DNA (H2A) and a DNA remodeler protein (SNF2h) was examined. The experimental results were compared with random distributed signal positions within the same global structures. It could be shown that the real distributions are significantly different from the random ones. It is also possible to check the data for signal clusters and determine their properties. This was first tested on simulated distributions and later applied in the analysis of signal accumulations in biological structures like centromere protein clusters (CENP- A, CENP-B and CENP-C) within the human kinetochore. In addition, the axial position of the localization data within the structure can be determined with a precision of about 50nm. A model for the distribution of the fluorophores in tobacco mosaic virus structures was developed and fitted to the data. The width of these structures determined by electron microscopy to be 18nm could be confirmed. The mean localization accuracy in this case was 8nm. A new microscope setup was build to split the fluorescence signal emitted by the sample due to its different properties like direction of polarization or spectral range. Both parts of the signal could be imaged simultaneously on the same detector. This results in a shorter acquisition time for a two color measurement. Additionally, no mechanical drift between two measurements is apparent.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Bielefeld

Year

2011

Author dc:creator

• Gunkel, Manuel

Repository record source_url

https://pub.uni-bielefeld.de/record/2305089

OAI identifier oai:identifier

oai:pub.uni-bielefeld.de:2305089