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Improved technology for imaging living cells, specific cellular targets and organelles is having a dramatic impact on basic and applied research. By combining optical design and molecular genetics, a new series of tools is being developed and successfully applied together with classical probes. Novel labelling strategies, better software for image enhancement and analysis are now available and allow image acquisition with greater speed and precision. This lab manual, intended as for bench-top use, is suitable for both scientists and graduate students, combines an update on the most advanced imaging procedures with detailed protocols. Examples, cleverly selected from the wide repertoire of cell pyhsiology, cover different functional aspects such as distribution of multiple ions, electrical activity, exo-endocytosis, gene expression, and the cell cycle.