Swapping out specific chemical building blocks in fluorescent molecules called rhodamines can generate nearly any color scientists desire, Howard Hughes researchers reported yesterday in Nature Methods.
According to Luke Lavis, a group leader at Howard Hughes Medical Institute’s Janelia Research Campus, the new development offers scientists a way to adjust the properties of existing dyes deliberately, making them bolder, brighter, and more cell-permeable. This expanded palette of dyes could help researchers better illuminate the inner workings of cells, he says, adding that his team lit up cell nuclei, made larval fruit fly brains shine, and highlighted visual cortex neurons in mice.
Scientists used to concoct different dyes mostly by trial and error, Lavis says. "Now, we've figured out the rules, and we can make almost any color."
His team's method could allow chemists to synthesize hundreds of different colors, he says. Until recently, making new rhodamines wasn't easy. Scientists still boiled chemical ingredients in sulfuric acid. This forces the molecules to link together in a condensation reaction. Mixing in different building blocks can yield new and unusual dyes, but ingredients had to be tough enough to survive the boiling acid bath.
In 2011, Lavis's team developed a new way to tinker with rhodamines' structure, under milder conditions. Using a reaction sparked by the metal palladium, the researchers could skip the acid step and construct dyes with complicated building blocks. Four years later, the team revealed the Janelia Fluor dyes, fluorescent molecules up to 50 times brighter than other dyes, and more stable too. The secret behind the Janelia Fluor dyes is a tiny square-shaped appendage called an azetidine ring.
"For us, it was a total revolution in the field of single-molecule imaging," says molecular biologist Xavier Darzacq of the University of California, Berkeley. Before using the Janelia Fluor dyes, the fluorescent-tagged transcription factor proteins his team studied were too dim to capture in crisp images. Researchers had to hold the camera shutter open for 10 milliseconds to collect enough light. That's long enough for proteins to wander, so the image would come out blurry. But the Janelia dyes are bright enough that his team can capture molecules in action in just a millisecond, Darzacq says. Such quick snapshots have allowed his team to do lab experiments he describes as "simply unthinkable a few years ago."
Now, Lavis's group has figured out how to fine-tune their fluorescent dyes, by tweaking rhodamines' structure even further. Rhodamines have a basic four-ringed design with groups of atoms protruding from different parts of the rings. In previous work, the scientists developed strategies for coarse tuning dyes, snip out an entire appendage here, and you can make a green dye. Pop in a silicon atom there, and you've got red. Lavis discovered that by carefully placing just a few new atoms in the dye structure, the color and chemical properties of the dyes could also be fine-tuned, allowing many shades of green from a single scaffold.
"The key thing is that it's all modular and rational," Lavis says. Select the right atoms, he explains, and chemists can engineer dyes with nearly any property they want. The dyes are synthesized in a single step with inexpensive ingredients, which makes them cheaper than commercial alternatives, he adds.
Caption: Novel rhodamine dyes synthesized in the Lavis Lab fluorescing under UV illumination. Image courtesy of Jonathan B. Grimm