<p>Morphogenesis, the development of multicellular organisms, is controlled by morphogens, small signalling molecules, that form gradients across the embryo. While such gradients can emerge through several mechanisms, the establishment of stable gradients requires the reduction of morphogen diffusion by at least one order of magnitude, a process that has not been directly observed. Here, we develop Single-Plane Illumination Microscopy-based spatial Fluorescence Cross-Correlation Spectroscopy (SPIM- sFCCS) to directly measure the diffusion coefficient of the morphogen Squint in early zebrafish embryos as a function of topography and length scale. The diffusion coefficient of Squint is a function of its molecular size, the viscosity within the interstitial spaces, and possible crowding. Here, we show that beyond these general parameters, Squint’s diffusion coefficient changes on length scales that are commensurate with the diameter of the intercellular spaces in the embryo and that its slowdown is regulated by receptor binding, which is crucial for gradient formation and embryonic patterning.</p>