Development and application of software for analyzing advanced fluorescence spectroscopy data [thesis]

Waldemar Schrimpf
Fluorescence is a process that can be utilized in many different ways for scientific research. This potential and versatility has also been noticed by the Nobel Committee, which, in the last ten years, awarded two Nobel Prizes in Chemistry to the field, 2008 "for the discovery and development of the green fluorescent protein, GFP" [1] and just recently in 2014 "for the development of super-resolved fluorescence microscopy". [2] The former greatly extended the capabilities for specifically
more » ... specifically tagging individual proteins, opening the doors for the application of fluorescence microscopy and spectroscopy in studying biological systems. Super-resolution spectroscopy, on the other hand, covers less the preparation and more the analysis of a scientific experimentat. It is the most prominent representative of a wide variety of fluorescence based methods that have emerged in the last 20 years and were made possible by advancements in three different fields. The first includes developments in chemistry and biochemistry that provided fluorescence dyes with better and unique properties, and also new ways of attaching them to the analyte of interest in an efficient and specific manner. Another requirement was the improvement of instrumentation like excitation sources, optical elements, or detectors and other electronic devices. The third field involves advancements in new analysis methods, algorithms and software. Extracting the maximum amount of information from the data reveals more details about the investigated systems and results in a deeper understanding of their nature. This thesis puts a strong focus on this last aspect. It describes the development of software for analyzing advanced fluorescence methods and subsequently the implementation and application of these techniques. After a brief introduction in chapter 1, the concepts and methods relevant to this work are presented in chapters 2 and 3. Hereby, chapter 2 briefly describes the basics of fluorescence and microscopy in general and the instrumentation used in the other chapters. Chapter 3 than provides a quick overview of the covered methods, mainly focusing on fluorescence fluctuation and fluorescence lifetime techniques. The developed software called PIE Analysis with Matlab (PAM ) is introduced in chapter 4. PAM was designed as a platform for a multitude of advanced fluorescence analysis techniques, with a focus on combining them with pulsed interleaved excitation. The included methods can be used to analyze single point data (e.g. fluorescence correlation spectroscopy), images (e.g. image correlation spectroscopy), as well as fluorescence lifetime information (e.g. phasor fluorescence lifetime microscopy). Additionally, (PAM ) contains a simulation program allowing the user to generate data with known parameters, making it possible to test new methods and theories. The core concepts and focus of the central platform and the individual applications are presented, followed by a more detailed description of their main features and most important underlying algorithms. Chapter 5 focuses on the application of the presented techniques and software for studying metal-organic frameworks (MOFs). They constitute a group of materials i ABSTRACT formed by connecting inorganic building blocks via organic linkers to a three dimensional crystalline lattice. Due to their high porosity and relative surface area, MOFs show big potential for a variety of industrial applications, ranging from analyte sensing, over gas storage, to catalysis. Here, two different MOFs were investigated. The first one, MIL-101(Al)-NH 2 , is an aluminum based MOFs with free amino groups on the organic linker that can be used for posy-synthetic modifications, e.g. with fluorophores. Using correlative fluorescence lifetime and scanning electron microscopy, two quenching mechanisms of fluorescein bound to the MOF were identified. The first one is a photo electron transfer type quenching via unmodified amino groups, while the second mechanism depends on the particle morphology and is most likely caused by defects in the structure. In the second part of chapter 5, different methods for functionalizing the zirconium MOF UiO-67 were investigated using fluorescence lifetime microscopy. The analysis showed that de novo incorporation of large, complex functionalities during synthesis can introduce small defects to the crystal structure. Solvent assisted linker exchange, the second method investigated, showed less effect on the lattice. During this process, functional groups are bound in two distinct steps. The first involves a fast binding of the modified linker to under-coordinated metal sites at the crystal exterior. The much slower linker exchange only takes place at elevated temperatures. Since the rate limiting step is the substitution of a bound linker, the exchange is spatially homogeneous throughout the whole crystal. In chapter 6 the influence of chrome nano patterns on the diffusion in supported lipid bilayers is investigated. Since they are believed to completely halt mobility, chrome barriers are often used to divide lipid layers into individual, non-connected segments. We could show that, under the investigated conditions, diffusion across the barriers can still be maintained, albeit at a reduced rate. Detailed pair correlation spectroscopy and scanning electron microscopy analysis revealed no clear correlation between this mobility and barrier width or height. On the other hand, fluorescence recovery after photobleaching indicates the presence of a cutoff barrier thickness that determines whether diffusion is possible or not. ii
doi:10.5282/edoc.19708 fatcat:6t63jp2i2jcgbfioiuh2ltwmii