This chapter will attempt to provide an overview of the theory and details of some basic applications of the technique of confocal microscopy. Since this is intended to be a general introduction to the possibilities available, it is not appropriate to include detailed descriptions of the specialized techniques used in physiological studies leading to the imaging and measurement of Ca²⁺-containing samples. Instead, the reader is referred to Chapter 5 in this volume that deals specifically with these imaging techniques. In this chapter our aim is to define the properties of the instrumentation and to give a few examples of basic preparation techniques for defining structure and localizing target molecules in cells. As one example, however, we will refer to our simple imaging technique for studying the three-dimensional (3D) microscopic distribution of calcium during skeletogenesis in the mammalian fetus.