The use of fluorescent tags for in vivo tracking of proteins has provided an array of new data on cell function. Correspondingly, a variety of new methods utilizing these fluorescent tags have been developed. These methods must take into account all of the concerns of keeping live samples in conditions as close to physiological norms as possible, including temperature, CO2 levels, media composition, and reduction of phototoxic effects. The microscope itself should also be designed to maximize the benefits and minimize the risks inherent in these methods. We provide an overview of these concerns.
