SPT analysis

Single QDs were recognized by their diffraction-limited fluorescence spot shape and characteristic blinking and were tracked with a 50-ms time resolution. QD spatial coordinates were identified in each frame as sets of >4 connected pixels using a two-dimensional object (67). Continuous tracking between blinks was reconstructed for the particles that presented 2 to 3 pixels (0.32 to 0.48 μm) of maximal position change between two frames and maximal dark periods of 25 frames (1.25 s). MSD curves were calculated for reconnected trajectories of at least 20 frames (67). The QDs were considered synaptic if colocalized with PSD-95 dendritic clusters for at least five frames. Instantaneous diffusion coefficients, D, were calculated from linear fits of the first four values of the MSD versus time plot according to the following equation: MSD(t) = <r²> (t) = 4Dt. The resolution limit for diffusion was 0.0075 μm²/s (67), whereas the resolution precision was ∼40 nm. The diffusive properties of the mobile receptor population were described as their median ± interquartile range (IQR), defined as the interval between the 25th and 75th percentiles. The software used in QD tracking was provided by D. Choquet (Bordeaux, France)