Fast and Precise 3D Fluorophore Localization based on Gradient Fitting

Astigmatism imaging approach has been widely used to encode the fluorophore’s 3D position in single-particle tracking and super-resolution localization microscopy. Here, we present a new high-speed localization algorithm based on gradient fitting to precisely decode the 3D subpixel position of the fluorophore. This algebraic algorithm determines the center of the fluorescent emitter by finding the position with the best-fit gradient direction distribution to the measured point spread function (PSF), and can retrieve the 3D subpixel position of the fluorophore in a single iteration. Through numerical simulation and experiments with mammalian cells, we demonstrate that our algorithm yields comparable localization precision to the traditional iterative Gaussian function fitting (GF) based method, while exhibits over two orders-of-magnitude faster execution speed. Our algorithm is a promising high-speed analyzing method for 3D particle tracking and super-resolution localization microscopy.