Since the discovery of GFP over 50 years ago, the growing spectrum of fluorescent proteins (FPs) has been an invaluable resource for studying the organization and function of cellular systems. FPs have been used to track protein localization, cell structure, intracellular trafficking, and protein turnover rates. Additionally, by engineering FP fusions associated with cellular organelles, scientists have been able to study many cellular processes, including mitosis, mitochondrial fission/fusion, nuclear import, and neuronal trafficking. Although FPs have enabled discovery of many cellular mechanisms, there are some limitations to working with FPs. Overexpression of fluorescently tagged proteins can lead to improper protein localization, protein aggregation, or disruption of normal protein function, and ultimately misinterpretation of the protein’s cellular role.