The image-Mean Square Displacement technique (iMSD) applies the calculation of the mean square displacement (MSD) used in single molecule tracking to images without resolving single particles. The iMSD plot obtained is similar to the MSD plot obtained using the single-particle tracking technique and is used to reconstruct the protein diffusion law. From these plots, we can determine whether the labeled molecules are undergoing pure isotropic, restricted, corralled, transiently confined or directed diffusion. In our study total internal reflection fluorescence (TIRF) microscopy images were taken of Cholera toxin subunit B (CtxB) membrane labeled cells. We mapped the modes of CTxB diffusion when bound to GM1 in live cell membranes and found different types of diffusion across the cell including isotropic, confined and transiently confined. However, no directed diffusion could be observed. We show that repeated illumination of the same area of the cell leads to a transition of diffusion type from confined to isotropic diffusion mode, highlighting a potential artefact that can be produced by performing FRAP experiments to investigate diffusion processes at the cell membrane. We also combined the iMSD analysis with number and brightness analysis to evaluate the influence of cluster formation on the diffusion of CTxB/GM1. We find that CTxB/GM1 are binding to immobile structures or are incorporated in areas of extremely slow diffusion in the cell membrane and that CTxB/GM1 are dynamically partitioning in membrane nanodomains as small as 60 nm in diameters. We show that the formation of clusters favors isotropic diffusion and reduces the rate of diffusion. We also report that in confined diffusion regime we observe a large range of diffusion rates demonstrating that the size of the confinement does not determine the diffusion rates and suggesting that the mechanism of confinement is different from the mechanism controlling CtxB/GM1’s diffusion rates.