The optical microscope is a powerful instrument for observing cellular events. Recently, the increased use of microscopy in quantitative biological research, including single molecule microscopy, has generated significant interest in determining the performance limits of an optical microscope. Here, we formulate this problem in the context of a parameter estimation approach in which the acquired imaging data is modeled as a spatio-temporal stochastic process. We derive formulations of the Fisher information matrix for models that allow both stationary and moving objects. The effects of background signal, detector size, pixelation and noise sources are also considered. Further, formulations are given that allow the study of defocused objects. Applications are discussed for the special case of the estimation of the location of objects, especially single molecules. Specific emphasis is placed on the derivation of conditions that guarantee block diagonal or diagonal Fisher information matrices.