Emulating single fluorophores with fluorescent beads

Precise calibration is essential in advanced single-molecule microscopy for accurately measuring fluorophore orientation and localization. Fluorescent beads are commonly used to calibrate and characterize system aberrations and polarization distortions. However, their larger size relative to single fluorophores, along with their unpolarized emission, results in a point spread function (PSF) that does not fully capture the behavior of a less wobbly single fluorophore. This study employs numerical and semi-analytical methods to evaluate how well a fluorescent bead of a given radius, within a polarizer-assisted microscopy setup, can emulate the properties of a single fluorescent molecule.