Advanced light microscopy and reporter gene techniques are indispensable for mechanistic studies on the functioning and organization of cells. New microscopy technologies and digital imaging allow us to study dynamic processes at shorter time scales and with higher spatial resolution in living cells. In this practical course the principles and cell biological application of different microscopy techniques will be taught. Topics include microscope construction, (green) fluorescent proteins (GFP), probes for measurement of cellular physiology, widefield and confocal fluorescence microscopy, differential interference contrast microscopy (DIC), fluorescence lifetime imaging microscopy (FLIM), Förster resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP), photoconversion, superresolution microscopy, digital image analysis and image noise. These techniques and concepts will be applied to study (sub)cellular physiology; dynamics of membrane organization; (intercellular) protein diffusion; hormonal signalling and intermolecular interactions. An important aspect of the course is data acquisition, reduction, processing and analysis; How can quantitative data be extracted from digital images for comparison with quantitative models of cellular mechanisms?
