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A fluorescence correlation spectroscopy (FCS) system based on two independentmeasurement volumes is presented. The optical setup and data acquisitionhardware are detailed, as well as a complete protocol to control the location,size and shape of the measurement volumes. A method that allows to monitorindependently the excitation and collection efficiency distribution isproposed. Finally, a few examples of measurements that exploit the two spots instatic and/or scanning schemes, are reported.

A versatile dual spot laser scanning confocal microscopy system for advanced fluorescence correlation spectroscopy analysis in living cell