Three dimensional (3D)microscopy is a powerful toll for the visualization of biological specimens and processes. In 3D microscopy, a 3D image is collected by recording a series of two-dimensional (2D) images focusing the microscope at different planes through the specimen. Each 2D optical slice in this through focus series contains the in-focus information plus contributions from out-of-focus structures that obscure the image and reduce its contrast. There are two complementary approaches to reduce or ameliorate the effects of the out-of-focus contributions, optical and computational. In the optical approach a microscope is used that avoids collecting out-of-focus light, such as a confocal microscope (see and references therein), a two-photon or three-photon fluorescence excitation microscope, or atwo-sided microscope. In the computational approach, the through-focus series is processed in a computer using any of a number of debluring algorithms to reduce or ameliorate the out-of-focus contributions. In the past two decades, several methods for debluring microscopic images have been developed whose common aim is to undo the degradations introduced by the process of image formation and recording