Hochdurchsatz-Fluoreszenz-Weitfeld-Mikroskopsystem
Final Report Abstract
Since the beginning of its operation, the microscope has been extensively used for automatic fluorescence image acquisition both in living cells and in fixed samples in S. cerevisiae and E. coli. We established protocols for high-throughput image acquisition (multiposition, multicolor and z-stacks) in multiwell plates and for fluorescence quantification in single cells. Using these protocols, images from an whole library of 5500 yeast knock-out strains could be acquired. In S. cerevisiae, the instrument was mainly used to follow the response to a range of chemical stimuli and environmental perturbation of the MAP kinase network by imaging several fluorescence reporter genes in living cells. The acquisition was done in a parallelized manner, acquiring measurements from different strains and comparing different levels of stimulation. The motorized stage's precision and speed were crucial for this task, since they determine the time resolution, and therefore the quality of the data set used for the subsequent analysis. In E. coli, the automated imaging was used to follow formation of biofilms, investigating changes in single-cell gene expression in time- and space-resolved manner. The instrument was also extensively used for establishing an automated approach for measuring protein-protein interactions by acceptor photobleaching Förster Resonance Energy Transfer (FRET), applicable both in S. cerevisiae and In E. coli. Here, the ability of controlling every single microscope component (motorized stage, shutters, fluorescence light attenuator) via software was crucial in obtaining reproducible data and allowed to significantly reduce the measurement times. In yeast, protein-protein interactions in the spindle positioning checkpoint and in the MAP kinase network were mapped. In bacteria, FRET measurements contributed to the identification of SiiA and SiiB as novel type I secretion system subunits controlling SPI4-mediated adhesion of Salmonella enterica. Currently, the system is used to perform an automated screen of interactions among numerous E. coli membrane chemoreceptors.
Publications
- SiiA and SüB are novel type 1 secretion system subunits controlling SPI4-mediated adhesion of Salmonella enterica. Cell Microbiol. 2014
Wille T., Wagner C., Mittelstadt W., Blank K., Sommer E., Malengo G., Döhler D., Lange A., Sourjik V., Hensel M., Gerlach R.G.
(See online at https://doi.org/10.1111/cmi.12222)
