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Confocal laser scanning microscopy

Confocal laser scanning microscopy enables imaging at high optical resolution and contrast. By collecting sets of thin optical slices at different depths within a thick object the three-dimensional structure of the latter can be reconstructed. We use a Leica SP5II setup equipped with 63x oil and glycerol objectives for high resolution imaging and with blue, green, red and far red laser lines.

Fluorescence resonance energy transfer microscopy

Fluorescence or Förster resonance energy transfer (FRET) is based on the energy transfer between two light-sensitive molecules. We use this method to investigate protein-protein interactions (intermolecular FRET) as well as conformational changes within one molecule (intramolecular FRET) in real-time and in living cells or tissue slices. The respective proteins are labeled with two spectrally distinct fluorophores, one serving as the ‘donor’ transferring the excitation energy to the other called the ‘acceptor’. This energy transfer largely depends on the distance between them, which means FRET can be used as a very sensitive molecular ruler We record the fluorescence of donor and acceptor molecules using a Zeiss Axioobserver Z1 epifluorescence microscope equipped with DualView2 and Evolve camera.