Fast STED Microscopy

Abstract

The spatial resolution of optical far-field microscopes was hampered by diffraction until STED microscopy broke the diffraction limit. This thesis for the first time combines the high spatial resolution of STED microscopy with high temporal resolution to Fast STED microscopy. It heralds the study of fast dynamic processes via STED microscopy: diffraction unlimited movies with 200 images per second are recorded from colloids; video-rate imaging is achieved in living cells. In particular, the diffusion of 36 nm particles is shown at 80 frames per second (fps); the processes at the growth front of a colloidal crystal of particles as small as 200 nm are visualized at 200 fps. Biological samples are imaged and analyzed beyond the proof of principle, driven by open questions from actual biological research: it is shown that the movement of neurotransmitter vesicles encompasses low and high mobility states, and that young and mature vesicles show a distinct motion behavior. The use of pulsed and continuous wave lasers is compared for Fast STED microscopy of living neurons. Customized solutions for the quantitative analysis of the movies, especially for the localization and tracking of neurotransmitter vesicles, are developed and tested in Monte Carlo simulations. The algorithms are extended to show the correlation of the metabolic rate of cells with the density of the Translocase of the Outer Membrane in mitochondria. Beam-scanning STED microscopy is extended to two-color imaging and protein interactions in human stem cells are therewith analyzed, which introduces high-resolution imaging into medical research. The co-localization of the proteins CD63, TIMP-1 and β1-Integrin is shown. The thesis also presents the historic evolution of the knowledge about the diffraction limit and discusses the influence of noise and pixilation on resolution. STED-Mikroskopie wird auf Zweifarbenaufnahmen ausgedehnt. Damit werden Proteininteraktionen in menschlichen Stammzellen untersucht; dies f ührt die optische hochauflösende Bildgebung auch in die medizinische Forschung ein. Die Kolokalisierung der Proteine CD63, TIMP-1 und β1-Integrin wird nachgewiesen. Diese Doktorarbeit enth ält auch einen historischen Abriß der Erkenntnisse zur Beugungsbegrenzung und diskutiert den Einfluß von Rauschen und Pixeln auf die Auflösung.