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The eye is outstanding in its sensitivity: it can react to single photons due to the rod photoreceptor, in particular its outer segment. This thesis investigates experimental approaches to study the higher-order structure of the outer segment’s crucial constituent: rhodopsin. The oligomeric state and organization of rhodopsin within disk membranes has been discussed intensively due to its fundamental meaning. The method of choice to analyze rhodopsin in its close-to-native state so far has been cryo-electron microscopy of vitreous sections (CEMOVIS). To analyze one illumination state of rhodopsin (dark state), CEMOVIS was sufficient. However, CEMOVIS has the disadvantage to be time-consuming and error-prone. To analyze different illumination states, CEMOVIS is not efficient enough. The main part of this thesis treats alternative techniques to overcome the disadvantages of CEMOVIS. Additionally, studies of rhodopsin’s temporal bleaching behaviour were carried out. In more detail: First, we aim to analyze the bleaching of intact retinas. This was done quantitatively via UV-VIS-spectroscopy. We could see an exponential bleaching within the short time scale (in the range of ms). Second, alternative techniques to substitute CEMOVIS were considered and their applicability investigated and evaluated. Dual-axis tomography for tomogram acquisition of samples produced via CEMOVIS turned out to be suboptimal. A ROS solubilization prior to cryo-fixation was accompanied by the same disadvantages like CEMOVIS of high-pressure-frozen retinas. The retinal misplacement and thereby the oblique fractioning of ROS during freeze-fracture experiments with retinal cryo-samples impeded direct imaging of exposed ROS disk membranes. Samples for cryo-ET can also be produced with the focused-ion beam (FIB). Tomography is sped up by using the Fib , due to superior sample quality. However, this way to produce samples is again suboptimal and time-consuming at the present. The most promising approach was to reduce dimensionality of the sample via isolation of ROS disks. ROS disks can subsequently be used for vitrification (plunge-freezing) and subsequent cryo-ET or analyzed via atomic force microscopy (AFM). AFM however, does not allow the investigaion of close-to-native state samples. Nevertheless, it has the advantage of being a technique with a greater effectiveness – what we aimed for. A future optimization of this technique will allow for further investigations of, for example, rhodopsin’s supramolecular structure upon illumination. Finally, a tomogram was simulated and compared to real data. |
