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Over the past decade, microfluidic techniques have been established as a versatile platform to perform live cell experiments under well-controlled conditions. To investigate the directional responses of cells, stable concentration profiles of chemotactic factors can be generated in microfluidic gradient mixers that provide a high degree of spatial control. However, the times for built-up and switching of gradient profiles are in general too slow to resolve the intracellular protein translocation events of directional sensing of eukaryotes. Here, we review an example of a conventional microfluidic gradient mixer as well as the novel flow photolysis technique that achieves an increased temporal resolution by combining the photo-activation of caged compounds with the advantages of microfluidic chambers.