The use of super-resolution imaging techniques, such as PALM and STORM, for studying cellular ultrastructures relies on photoactivatable fluorescent proteins (PAFPs). Some characteristics that are key to the performance of these PAFPs include size, brightness, oligomeric state and stability. EosFP is the best overall green-to-red PAFP available, but because it is inherently tetrameric, derivative monomeric and dimeric forms have been generated, with the most widely used being mEos2. mEos2 is limited, however, by its tendency to oligomerize at high concentrations, which is worse when it is used to label membrane proteins. Indeed, Pingyong Xu, Tao Xu and colleagues found that mEos2 fused to Orai1, a subunit of the calcium release–activated calcium (CRAC) channel, or to glucose transporter 4, causing formation of protein aggregates. In an effort to generate a stable monomeric PAFP, the authors first solved the crystal structure of mEos2 to determine the molecular basis for its oligomerization. The mEos2 structure, a tetramer comprised of four β-cans, revealed three key residues that contribute to oligomerization. Mutation of two of these residues eliminated mEos2's tendency to oligomerize, but the mutant was substantially less bright than the original protein. To enhance brightness, the authors then made additional mutations near the chromophore, resulting in two variants called mEos3.1 and mEos3.2 that are brighter in the green form than mEos2. Orai1 fusions with mEos3s correctly localized to the plasma membrane and exhibited large CRAC currents, indicating that the fusion interfered minimally with channel function. Additional fusions to other membrane proteins and intracellular markers also showed the expected localization, further demonstrating that the mEos3s behave as good fusion partners. mEos3.2 had the best characteristics overall for green-to-red PAFPs in terms of brightness, stability, monomericity and label density, making it a viable probe for super-resolution imaging studies. (Nat. Methods doi:10.1038/nmeth.2021, published online 13 May 2012)