A new molecule capable of emitting the brightest turquoise light to date could improve imaging sensitivity in living cells. First obtained in 1994, cyan fluorescent proteins (CFPs), when attached to an active protein, allow mapping of many processes in living cells. When a cell is illuminated with blue light, CFP is induced to emit its characteristic cyan color. The earlier molecules, however, demonstrated a weak fluorescence level, converting only 36 percent of the incoming blue light into cyan light. “That means only one out of three photons absorbed by the chromophore is re-emitted as a fluorescent photon,” Antoine Royant of the Institut de Biologie Structurale in Grenoble told BioPhotonics. “Since 2004, there had been sequential improvements in CFPs by tedious trial-and-error approaches, progressively increasing the quantum yield to 49, 56 and 84 percent.” By understanding how the various mutations contributed to the increased quantum yield, Royant and scientists from the European Synchrotron Radiation Facility (ESRF), also in Grenoble, and the universities of Amsterdam in the Netherlands and Oxford in the UK hypothesized that the protein could be further improved by optimizing the interactions between the chromophore and the surrounding protein at a specific location. Using their new method, the team achieved higher brightness and, with it, enhanced fluorescence imaging sensitivity. “By using the innovative screening technique of our collaborators, we could design a much improved CFP, mTurquoise2, whose quantum yield of 93 percent is very close [to] the ideal 100 percent,” Royant said. A tiny crystal of mTurquoise2 viewed with a microscope. The crystals were used to study the atomic-scale interactions that result in mTurquoise2’s high fluorescence efficiency. Courtesy of von Stetten/Royant/CNRS-ESRF. The quantum yield of the first usable CFP, or ECFP (enhanced cyan fluorescent protein), was limited to 36 percent because the molecule suffered from a high level of structural dynamics in the vicinity of the chromophore, Royant said. “Part of the protein undergoes relatively large-scale movements, coming into contact with the chromophore from time to time on a nanosecond timescale, causing occasionally nonradiative de-excitation of the excited chromophore.” Known as collisional fluorescence quenching, this flaw in CFPs was progressively suppressed with improved mutations. Fine-tuning of the interactions between the chromophore and the protein enabled the scientists to achieve the 93 percent quantum yield. At ESRF, the Oxford and Grenoble teams detected subtle details on 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 the chromophore region, the structures showed a subtle process. “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,” David von Stetten of ESRF said in a press release. The new molecule is more sensitive than any other CFP because it needs 20 percent fewer photons of blue light than a cell labeled with the best previous CFP to obtain the same signal. “Decreasing the amount of light to get a signal is vital because fluorescent proteins eventually bleach in a nonreversible manner, which limits the span of an experiment,” Royant told BioPhotonics. “The less light is needed, the longer the experiment can last. This is crucial if you are looking at cellular processes on several hours’ timescale.” The turquoise protein is suitable for two main applications in cell biology. Scientists can image subcellular compartments such as mitochondria, nuclei and various membranes by colocalizing any protein of interest by fusing its DNA sequence with a fluorescent protein. “The cyan color of CFPs will be useful for multicolor labeling when one wants to visualize, let’s say, four proteins or compartments at the same time,” he said. It also could be used in conjunction with a yellow fluorescent protein (YFP) for Förster resonance energy transfer applications to analyze protein-protein interactions in living cells with maximum sensitivity. “While this new protein does not allow for completely new experiments, it can easily be used to replace inferior CFPs in existing cell biology constructs, enabling better results due to an improved signal-to-noise ratio,” Royant said, adding that it pushes the limits of what is possible by one significant step. Next, the team plans to design improved fluorescent proteins that emit light in various colors for use in other applications. “Yellow fluorescent protein would be the obvious next step because it is the other half of the popular Förster resonance energy transfer tandem YFP-CFP, and can still be improved in terms of brightness,” he said. “Besides, red fluorescent proteins have lower quantum yields than cyan, green, yellow and orange fluorescent proteins and, thus, could be improved as well. “Our research is primarily understanding the relations between structure and spectroscopic properties of all kinds of proteins which are colored. That means solving high-resolution structures as well as measuring absorption, fluorescence or Raman spectra on the protein crystals themselves.” The research was published March 20 in Nature Communications (doi: 10.1038/ncomms1738).