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978-3-8439-1498-7, Reihe Physikalische Chemie
Advanced Confocal Microscopy: From Setups To Applications
168 Seiten, Dissertation Ludwig-Maximilians-Universität München (2014), Softcover, A5
Confocal microscopy is known for its capability to produce exceptional 3D images, even in living tissue. At the same time, it is a powerful spectroscopic tool. In this work, I will describe the assembly of two instruments: The first is a multi-parameter fluorescence detection (MFD) setup. It is a spectroscopic tool that is able to characterize a fluorescent molecule, delivering information like fluorescence lifetime, anisotropy or the speed of its diffusion. The second instrument, a stimulated emission depletion setup, enhances the resolution capability of a confocal microscope. One particular problem of fluorescent microscopes, though, is that image resolution is always restricted to the diffraction limit of the wavelength of the laser light. The STED setup utilizes the effect of stimulated emission to circumvent the diffraction barrier and allows images with a three-fold resolution increase, down to 75nm.
These two setups will be used for several applications: The first will be centered around the molecular conformation of proteins, which are sensitive to the nature of the aqueous environment. In particular, the presence of ions can stabilize or destabilize (denature) protein secondary structure. I will apply single-pair FRET to a small 29 amino acid long model peptide to investigate unfolding mechanisms of different unfolding reagents from the Hofmeister series. The results show that certain salts achieve the unfolding by either collapsing the molecule to a compressed state or swelling it to a denatured state.
The second application of the MFD setup is the investigation of the enhanced green fluorescent protein (EGFP). It is still not fully known what percentage of EGFP is fluorescent. This lack of knowledge makes it nearly impossible to make quantitative statements. With the help of FCCS, it is shown that the folding efficiencies range from 40 − 90%, depending on the environment of the fluorescent protein and which particular mutant is used.
In the third application, the focus will be shifted to nucleation- and polymerization- behavior of actin. In order to compare results of confocal spectroscopy methods with well-established bulk essays, we successfully ported the standard bulk essay to the confocal microscope, allowing for the first time to follow the decrease of monomer concentration and appearance of small filaments. Also, the formation of dimers or other small oligomers below the critical concentration is proven for the first time, using FCCS.