Live cell imaging provides a powerful technique for the analysis of molecular dynamics within cells. Advances in imaging technology and probe design have established this approach as an important tool in modern biology. It is now possible to obtain commercial turnkey systems for digital imaging using a number of different imaging modalities. Nevertheless, it still requires considerable technical care and expertise to conduct a successful experiment. To perform a successful imaging experiment, it is important to maximize the signal-to-noise ratio (S:N) while minimizing damage to the cells. In this article, we focus on the use of fluorescence microscopy in live cell imaging, although most of the points discussed are relevant to any type of imaging. We describe many of the methods and considerations that are required for performing a successful imaging experiment in living cells. However, we do not provide a single recipe for success: The approach is much too empirical and depends on careful observation of the particular cells under investigation.