Caged compounds time resolution

Quantitative studies of chemotactic signaling require experimental techniques that can expose single cells to chemical stimuli with high resolution in both space and time. Recently, we have introduced the method of flow photolysis (Anal. Chem. 79 3940-3944, 2007), which combines microfluidic techniques with the photochemical release of caged compounds. This method allows us to tailor chemical stimuli on the length scale of individual cells with subsecond temporal resolution. In this chapter, we provide a detailed protocol for the setup of flow photolysis experiments and exemplify this versatile approach by initiating membrane translocation of fluorescent fusion proteins in chemotactic Dictyostdium discoideum cells. 

The time resolution of the photochemical release of caged substrates depends on two factors the energy/time profile of the light source used for photolysis and the effective rate of liberation of active substrate. It should be remembered that the time scale of the potential application of a kinetic technique depends critically on the accuracy of the determination of zero time. Some practical details of the investigation of different parameters are illustrated below. Suffice it to say for the moment that the range of compounds presently available for different systems have time constants from 10 ms to 10 ps for the liberation of phosphates and amino derivatives (neurotransmitters). 

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