Computational modeling of endocytic vesicle formation imaged by simultaneous two-wavelength axial ratiometry

Clathrin-mediated endocytosis (CME) facilitates the internalization of extracellular cargoes. However, how clathrin-coated vesicles (CCVs) form remains unclear due to the limited resolution of live-cell fluorescence microscopy and the need for sample fixation in electron microscopy. To bridge this gap, our lab developed Simultaneous Two-wavelength Axial Ratiometry (STAR) microscopy that leverages the wavelength-dependent properties of Total Internal Reflection Fluorescence (TIRF) microscopy. Dual-tagging a protein of interest with two spectrally separated fluorophores allows STAR to measure the intensity ratio and retrieve the z-position of the protein. Although the exponential decay of the evanescent wave is critical for STAR, it results in an uneven excitation of fluorescently tagged proteins. This will bias STAR measurements when dual-tagged proteins are distributed on a 3D object such as CCVs. To understand the accuracy of STAR for studying vesicle formation, we used mathematical modeling. We represented the CCV as a monodisperse sphere and used the STAR equations to calculate vesicle height (∆z) and compared it to the “ground truth” center of mass (CM). We investigated the influence of vesicle formation, radius, the number and distribution of proteins, the distance of the vesicle from the plasma membrane, and Poisson noise on STAR. This mathematical model of STAR microscopy assesses how the ∆z measured by STAR compares to the ground truth. The theoretical findings will be used to relate experimental ∆z results to vesicle morphology and eventually to develop a 3D dynamic model of CCV formation from our experimental data.