A real time 2D deconvolution system and method for processing a time sequence, or video sequence, of microscopy images. A system is provided that includes a first algorithm for processing a first image in the set of 2D images, wherein the first algorithm calculates an estimated point spread function (PSF) by analyzing data in the first image; and a second algorithm for processing a set of subsequent images in the set of 2D images, wherein each subsequent image is processed based on the estimated PSF, under the assumption of slowly-varying image change. A PSF refinement system may optionally improve the PSF estimate, between each image, as computation time permits.