Since its introduction in 1983, deconvolution microscopy has become a key image-processing tool for visualizing the cellular structures of fixed and living specimens in three dimensions and at subresolution scale. The last 20 years have seen the development of many different applications based on deconvolution microscopy, including a wide variety of optical setup and deconvolution algorithms. This chapter aims to summarize and to describe in detail the major features of this technology, from theoretical aspects to practical solutions. It will begin by explaining the principle of image formation in three-dimensional optical sectioning microscopy. As deconvolution microscopy provides, in essence, a means of overcoming the limits of optical microscopy, the second part of this chapter is dedicated to the theoretical and experimental description of image generation through a microscope. Methods will be detailed for the determination of point spread function, as a crucial step for the characterization of any optical system and a key preliminary step for image deconvolution. The challenges faced and the various possibilities for determining this function precisely will be discussed. All possible sources of aberrations and image degradation processes will be discussed. In the third part of this chapter, we will introduce the acquisition setup and requirements for compliance between acquisition and deconvolution processes. Typical setups for fixed and living cell observation will be detailed, with key features for optimizing speed and reducing artifacts. In the fourth and last part of this chapter, we will describe, in theoretical terms, the various restoration algorithms commonly used in the field of optical microscopy and will provide results obtained with some of the commercially available packages. We shall conclude by considering the prospects for future solutions (currently under development) aiming to handle more easily the huge amounts of data generated by rapid multi-dimensional living cell microscopy. Designed for use by standard cell biologists and hardware and software engineers and developers, this chapter has been written to provide a clear explanation of the wide-reaching and powerful domain of deconvolution microscopy.