Researchers have developed a simple method for making fluorescent dyes of many colors that can be activated by visible light. The method involves switching out a single oxygen atom in existing fluorescent dyes for a sulfur atom, which makes them photoactivatable. The approach provides a new set of tools for visualizing how proteins and other […]
Researchers have developed a simple method for making fluorescent dyes of many colors that can be activated by visible light. The method involves switching out a single oxygen atom in existing fluorescent dyes for a sulfur atom, which makes them photoactivatable. The approach provides a new set of tools for visualizing how proteins and other molecules behave in living cells without damaging tissue.
Today’s light microscopes are so powerful that they can track molecules at nanometer-scale resolution as a cell conducts its business. Researchers want to be able to turn fluorescent dyes on and off in order to selectively light up and track specific molecules over time in living cells. But scientists have struggled to capture these close-ups because the photoactivatable dyes commonly used for labeling molecules have some major limitations: the dyes can be bulky, so they can’t label smaller molecules with good resolution, and they need to be activated with high-energy laser light. The intense illumination damages DNA and mitochondria and causes proteins to cross-link. It also bleaches the fluorescence that the dyes deliver. So dyes that can be triggered with low-power visible light would be a boon for biological imaging in living tissue.
Previous work had shown that trading the carbonyl group for a thiocarbonyl group in a dye can quench fluorescence nearby. In the new study, Han Xiao, a chemist and bioengineer at Rice University, and his team demonstrated that if the thiocarbonyl group is incorporated into the conjugated system of an ordinary, nonphotoactivatable fluorescent dye, the dye can be photo-oxidized with visible light. Xiao and his team used a one-step chemical reaction involving a thionating agent called Lawesson’s reagent to switch the carbonyl group’s oxygen to sulfur, essentially suppressing the glow.
Credit: J. Am. Chem. Soc. A photoactivated red dye (bottom left) labels lipid droplets in a cell just like a commercial nonphotoactivatable green dye (top right). Brown shows the colocalization of the dyes (bottom right). Cell nuclei are stained blue.
Unleashing the fluorescence simply involves flashing a dye solution with low-intensity visible light of a wavelength the same or shorter than that emitted by the dye, Xiao explains. If the dye is red, for example, a red, green, or blue light would activate it. “The chemistry we are using is really simple,” Xiao says. The photoactivation requires just visible light and oxygen, which is present at sufficient levels naturally dissolved in saline buffer.
Xiao’s team used the approach to create light-activated versions of common red, green, blue, and purple dyes that can be made to target different biomolecules or parts of the cell. They used one of the dyes to visualize lipid droplets and another to mark specific sites within a protein. They also demonstrated that they could use two different dyes simultaneously to label different parts of a lipid droplet.
Luke D. Lavis, a biochemist at Janelia Research Campus, says he likes the paper because the fluorescence quenching is accomplished with substitution of a single atom, so the resulting molecule is small. Researchers could encode such a molecule into a protein that could then be made to fluoresce without using any additional reagents.
A huge variety of nonphotoactivatable dyes exist, and most of them should respond to this strategy, Xiao says. “The beauty of our chemistry is that you don’t need a really strong light source. You can use an LED you get from Amazon, and it works.”
Xiao’s team is continuing to make more dyes using the approach. They are also testing the strategy for near-infrared dyes, which can penetrate tissue more deeply.
This article is reproduced with permission from C&EN (© American Chemical Society). The article was first published on September 30, 2019.