Figure 1: iMSD analysis of molecular motion in 3D: GFP Brownian diffusion in dilute solution.

(a) Pictured experiment: ‘fluorescent’ molecules freely diffuse distributing in space and time. Scanning the samples with decreasing speed allows measuring the average particle displacements over a wide spatiotemporal scale. (b) When the scanning is faster than the particle dynamics, the particle image corresponds to the autoconvolution of the instrumental point spread function, which is well approximated by a Gaussian profile. On the other hand, when scanning speed decreases, the particle starts to move significantly, that is, the correlation function squeezes in space becoming much larger than the PSF. Equation 15 in Supplementary Information describes this deformation effect as a function of the particle average displacement, and allows recovering the iMSD as a function of time delay. (c) Schematic representation of the iMSD for a molecule diffusing in 3D.