A molecule capable of emitting turquoise light within living cells three times brighter than ever achieved could improve the sensitivity of cellular imaging. Cyan fluorescent proteins (CFPs), when attached to an active protein, can span several processes in living cells. By illuminating a cell with blue light, the concentration of CFP remains within the cell, stimulating the fluorescent protein to emit a significant cyan color. However, these molecules demonstrate a weak fluorescence level, converting only 36 percent of the incoming blue light into cyan light. An artistically inspired visualization of the 3-D x-ray structure of the cyan fluorescent protein mTurquoise2. (Image: Nature Communications/von Stetten/Royant/Goedhart) Now, scientists from the European Synchrotron Radiation Facility (ESRF) and the universities of Amsterdam and Oxford have teamed with Antoine Royant from the Institut de Biologie Structurale in Grenoble to achieve higher brightness and, with it, enhanced fluorescence imaging sensitivity. At ESRF, the Oxford and Grenoble teams detected subtle details of how CFPs store incoming energy and retransmit it to fluorescent light using x-ray beams. As part of this initiative, tiny crystals of CFPs were created and their molecular structures determined. Near its chromophore region, the structures showed a subtle process. Fluorescence microscopy image showing the actin filaments in a living cell. These filaments play an important role in muscle contraction. Here mTurquoise2 proteins were fused to a small protein that attaches itself to the actin filaments. Thanks to the fluorescent light, vital process involving actin filament can be made visible in a living cell. (Image: Nature Communications./Goedhart) “We could understand the function of individual atoms within CFPs and pinpoint the part of the molecule that needed to be modified to increase the fluorescence yield,” said David von Stetten of ESRF. The Amsterdam team subsequently studied the properties of multiple CFP molecules, using innovative screening techniques. Their design resulted in a CFP called mTurquoise2. By integrating the structural and cellular biology processes, the scientists demonstrated that mTurquoise2 has 93 percent fluorescence efficiency. A tiny crystal of mTurquoise2 viewed with a microscope. The crystals were used to study the atomic scale interactions that result in mTurquoise’s high fluorescence efficiency. (Image: von Stetten/Royant/CNRS-ESRF) The new molecule will enable life scientists to analyze protein-protein interactions in living cells with maximum sensitivity. “Thanks to this novel approach taking into account the structural dynamics of the protein, scientists now hope to design improved fluorescent proteins emitting light of different colors for use in other applications,” Royant said. The research appeared in Nature Communications. For more information, visit: www.esrf.fr