Ds-SiFi: A Photo-Conversion-Based Approach to Quantify the Effective Diffusivity of Biomolecules <i>in vivo</i>

Currently, quantifying effective diffusivity in vivo is limited by the stringent assumptions, such as homogeneity, simplified geometry, steady state, etc., so that practitioners suffer from rough approximations and draw misleading conclusions or interpretations. Here, we report a general diffusivity quantification for biomolecules in reaction-diffusion systems with unprecedented accuracy and robustness. We demonstrate the first accurate measurement of morphogen effective diffusivity in highly dynamic and heterogeneous developing tissue with geometric constraints/irregularities. We find that identical morphogen molecules exhibit remarkable inter-tissue diffusivity differences of up to an order of magnitude. The robustness of a morphogen gradient and receptor influence on the gradient profile can be also evaluated by this new method we call Ds-SiFi. Ds-SiFi is further demonstrated to measure the diffusion coefficient of biomolecules intracellularly, in both the cytosol and plasma membrane. The novel insights provided by Ds-SiFi can transform our mechanistic understanding of diffusion-related processes.