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Biological processes show extreme complexity orchestrated both in space and time. To face this complexity, it has been necessary, as pointed out by R.Y. Tsien in his lecture "Unlocking Cell Secrets with Light Beams and Molecular Spies" (Heineken Prize for Biochemistry and Biophysics, 2002), to develop fluorescent spies in order to analyze, quantify in real time and decrypt very sophisticated cellular mechanisms. The recent explosion of fluorescent biosensors development opens a new field of investigation by allowing the analysis of extremely specific biological events at the level of cellular organelles, individual cells, tissues and even entire living organisms in physiological and pathological conditions. The development and the use of these tools require solving a number of conceptual, experimental and technological problems concerning both the biosensors themselves and the tools to follow them. Equally important, a range of new developments including optogenetic and optochemical tools allow systematic perturbation of biochemical processes while they are read out by optical biosensors. The purpose of this conference is to gather and consolidate the expertise in the field of biosensors and to promote the reflection and the exchange of information so as to accelerate the development of these new dynamic imaging approaches in all areas of biology and biomedical sciences.