You can never have enough photons.

Biologists, they always want more, says Luke Lavis, a dye chemist with Howard Hughes Medical Institute. And, he lets slip under a smile, they’re “super picky.”

Still, biologists appreciate his work. While Lavis’ fellow chemists might be inclined to see his latest series of molecular dyes as “incremental” advances, biologists are jumping at the chance to use them, he says. A dye ever-so-slightly brighter can make the difference between seeing a single protein’s true wiggly movements within a cell—illuminating hidden details of our basic biology—and seeing a useless, fluorescent blur.

The value of fluorescent dyes is not lost on commercial companies. Orders of dye intermediates and dyes similar to those Lavis makes can easily run into the tens or even hundreds of thousands of dollars. That can take a big chunk out of a researchers’ federal grants and budgets.

But Lavis—who notes he was raised by hippies in Oregon and doesn’t care about money—quietly shares his dyes for free with any researcher who reaches out to him. He posts his discoveries on bioRxiv—a pre-print server for life sciences manuscripts—and word spreads on Twitter, he says. Researchers in turn send him raw materials and, in the margins of his research days, he fills orders. Though Lavis says the dyes will one day be commercialized, he estimates he’s sent out millions of dollars-worth of free dyes to hundreds of labs around the world already. And his dyes have gone on to light up neurons in flies and mice. They’ve illuminated gene editing technology at work in cells, the intricate details of cell walls, and important stretches of DNA.

His latest dyes are his most popular yet.

Colorful chemistry

This week, Lavis and his colleagues reported in Nature Methods that they have come up with a tunable way to create bright, stable, cell-permeable dyes in any color a researcher could want.

The advance is an improvement on a class of dyes called “rhodamines.” Although his lab has been working on it for several years, he tells me, it’s an improvement that’s been in the making for over a century.

Sitting in an airy office overlooking a bucolic pond on HHMI’s Janelia Research Campus—a sprawling old farm property in Northern Virginia—Lavis takes me all the way back to the 1840s, when chemists started tooling around with coal tar.

B. Mole

B. Mole

B. Mole

Luke Lavis

B. Mole

B. Mole

B. Mole

Luke Lavis

“That’s when organic chemistry started,” he said. Chemists started purifying, distilling, and then experimenting with chemicals they found in coal tar, which they could purchase by the bucket. Not long after, chemists figured out that they could come up with useful—and valuable—textile dyes from coal tar, sometimes just by boiling things in strong acids.

In 1887, chemists first reported rhodamines. The orange-yellowish, small molecule dyes were bright and stable. But they had a big drawback: making rhodamines involved boiling them in sulfuric acid for days. Any chemistry needed to modify their backbone structures—say, chemistry needed to change the color or brightness of the dyes—had to survive the boiling. Despite their potential, rhodamines were of limited use to biologists for more than a century for this reason.

When green fluorescent protein (GFP) burst onto the biological scene in the 1990s, it quickly elbowed out dyes. GFP was easy to tack onto specific proteins so biologists could watch where they went and what they did. Still, GFP and the colorful derivatives that followed have drawbacks, too. They’re not that bright, and they’re cumbersome. If there’s too much GFP-tagged protein around in a cell, they tend to clump together. This ruins a researcher’s chances of seeing what the protein would normally be up to in the cell.

Researchers needed something brighter, nimbler.

Living color

Enter Lavis’ rhodamines. After boiling the dyes in lab for years—and ruining many pairs of pants with sulfuric acid splatter—Lavis figured out how to make the structures with much cleaner chemistry. He and fellow HHMI chemist Jonathan Grimm borrowed the Nobel-winning method of palladium-catalyzed coupling reactions. They started making un-boiled rhodamines. He and his colleagues published the method in 2011.

As biologists were pushing more and more for ways to tag and image single molecules in cells, Lavis saw potential for his chemistry. “We basically thought a lot about how to take one of these old rhodamine dyes and basically jazz it up to make it brighter and more photostable.”

So, they got to work making all sorts of new rhodamine dyes that would be useful for biologists. They tweaked side groups and added new features, like a chemical hitch to link them onto specific proteins. They also figured out that they could make the dyes brighter by adding four-member ring structures on the sides of the molecule. This kept the excited structure from relaxing back down to a ground state without emitting photons. In 2015, Lavis’ lab team revealed a set of rhodamine-based dyes that were more stable and 10 times brighter than the original rhodamine structure, plus less clunky than GFP.

Now, after trying out more chemical tricks, they’ve figured out how to tune rhodamine’s structure to create any color they want—across the whole range of the visible spectrum.

Researchers who ordered up Lavis’ dyes have been using them in fish, flies, and mice, as well as cells grown in lab. Lavis’ team is still fine-tuning to make them even brighter, stably tagged to proteins, and safe for all types of cells.

“We’re just chemists kind of goofing off in the lab,” he says, “but we get to work with a bunch of amazing biologists out there.”

Nature Methods, 2017. DOI: 10.1038/nmeth.4403 (About DOIs).