Imaging techniques to measure diffusion inside dendritic spines. (a) FRAP. Fluorescence intensity change is measured after photobleaching fluorescent molecules in a spine head. Fluorescence recovery rate is mostly determined by the exchange rate between spine and dendrite. (b) FCS. The fluctuation of fluorescence intensity from the detection volume fixed inside a spine head (blue region in left panel) is recorded as a function of time (center panel). Since the fluorescence intensity fluctuates as the molecules enter and leave the fixed detection volume, the characteristics of intensity fluctuation essentially contain information about local diffusion speed. To estimate the diffusion coefficient, the autocorrelation function of fluorescence intensity fluctuation is calculated (right panel). (c) SPT. In SPT, molecular trajectory is directly measured with video microscopy. To analyze the speed and pattern of molecular motion, mean squared displacement (MSD) is calculated. For diffusion without barrier, MSD increases linearly against time. On the other hand, for diffusion within the compartment, MSD converges to a certain value, which corresponds to compartment size. (d) Comparison of three measurement techniques.