Studying biological macromolecules in the natural environment of a living cell
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Light microscopy and fluorescence techniques allow examination of conformational and interaction dynamics of biological macromolecules in vitro, but also within individual cells, in some cases even at single molecule resolution. Light microscopy and fluorescence techniques permit the analysis of not only individual proteins but also functional macromolecular complexes, which have increasingly come into the focus of modern structural biology.
A wide range of light microscopy techniques can be applied from the determination of protein localisation in cells or tissue, analysis of protein interactions and dynamics to the tracking of dynamic processes in live environment.
Advanced light microscopy relies on the fluorescent labelling of the biological macromolecules of interest. This allows non-invasive imaging of proteins and nucleic acids in cells, tissues or living organisms, in three dimensions and possibly in multiple colours. A whole range of labelling strategies is available to the researcher. Fluorescent probes, which cover most of the visible part of the electromagnetic spectrum, include small organic fluorophores, fluorescent proteins and fluorescent nucleic acids. While being large in size, the genetically encoded fluorescent proteins have been very widely used. Further development led to a great number of available proteins with different spectral properties.
Methods like Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Energy Transfer (FRET) give insights into complex interactions of proteins which may be influenced by, for example, differences in subcellular localization or signalling events. Advanced light microscopy within an in vivo cellular context can ideally complement in vitro acquired atomic resolution functional information of proteins and protein complexes.
Users and staff will plan the experiments (e.g. microscope access) and organise the required reagents prior to the visit. The platform can provide expression vectors for different fluorescent proteins, fluorescence labels, and chemical reagents. At the facility users will be helped to produce material (e.g. protein expression in different cell lines for in vivo analysis, recombinant protein production for in vitro studies, chemical labelling of protein). Subsequently, they will use the appropriate microscopes (Fluorescent-Correlation Spectroscopy (FCS), Fluorescence Recovery After Photobleaching (FRAP), Fluorescence Energy Transfer (FRET), particle tracking and co-localisation). Users can collect fluorescence data, process and evaluate them using optimal equipment and software under the guidance of highly experienced light microscopists.