Cell-imaging and biophysical techniques have become integral components of basic biomedical research. These experimental approaches enable researchers to dissect dynamic biological pathways relevant to cell biology as a whole, and they have proven especially useful in elucidating the molecular mechanisms of diseased cells, such as those found in cancer. To help better examine these biological processes, we aim to develop a small, genetically encoded, high quality fluorescent tag for use in live-cell imaging and biophysical applications. We will combine structure-based design with precise biochemical analysis to identify probes that are compatible with the cellular machinery. Further steps will be taken to evaluate and implement the probes in living eukaryotic and mammalian cells. If successful, this technology would provide an alternative to the fluorescent proteins, which currently dominate the field, for a range of imaging and biophysical applications.