To better illuminate the inner workings of cells, researchers have developed a way to adjust the properties of fluorescent dyes deliberately, resulting in an expanded palette of dyes that are bolder, brighter and more cell-permeable. The novel technique could allow chemists to synthesize dyes in hundreds of different colors. For their work, researchers at Howard Hughes Medical Institute (HHMI) focused on rhodamines, a family of fluorescent dyes that are especially bright and cell-permeable, but somewhat limited in their color range. These are novel rhodamine dyes synthesized in the Lavis Lab. Courtesy of Jonathan B. Grimm. The HHMI team previously developed a way to manipulate the structure of rhodamines without the need to boil ingredients in a bath of sulfuric acid. This milder approach opened the way for the construction of dyes using more complicated ingredients than had been used before. The team developed the Janelia Fluor (JF) series of dyes — fluorescent molecules up to 50 times brighter than other dyes and offering more photostability as well. This image shows purifying a new Janelia Fluor 549 derivative using flash chromatography. Courtesy of Luke D. Lavis. To further refine the process, the team incorporated 3-substituted azetidine groups. This allowed the team to tune the spectral and chemical properties of the rhodamine dyes with unprecedented precision. It also allowed the team to establish principles for fine-tuning the properties of fluorophores and to develop a palette of new fluorescent and fluorogenic labels with excitation ranging from blue to the far-red. The researchers grafted different chemicals into the rhodamines and analyzed the new dyes’ properties. They demonstrated the versatility of the dyes in cell nuclei, tissues and animals. “The key thing is that it’s all modular and rational,” said researcher Luke Lavis. “No one had ever looked at rhodamines in this kind of systematic way before,” said researcher Jonathan Grimm. The dyes are synthesized in a single step using inexpensive ingredients, and cost just pennies per vial. The low cost has allowed the team to share its work with scientists around the world. The team has shipped thousands of vials to hundreds of different labs. “ These are novel rhodamine dyes synthesized in the Lavis Lab fluorescing under UV illumination. Courtesy of Jonathan B. Grimm. These dyes are a complete game-changer,” said Ethan Garner, a bacterial cell biologist at Harvard University who has used the dyes to trace the path of single molecules in his lab. According to Garner, the dyes developed by the HMMI team cover the entire spectral range. Scientists used to concoct different dyes mostly by trial and error, Lavis said. “Now, we've figured out the rules, and we can make almost any color.” The research was published in Nature Methods (doi:10.1038/nmeth.4403).