In Luke Lavis’s lab at HHMI’s Janelia Research Campus, scientists bring modern chemistry to bear on synthetic fluorescent dyes that have been around for a century and a half, tuning and tweaking them for specific biological applications. The infographic and brief story below guide you through the history of fluorescence – from discovery to the development of Janelia Fluor dyes.
If you’ve ever hung out in a darkened basement enjoying a gin and tonic while admiring a Grateful Dead poster under the subtle glow of a black light, then you’ve seen fluorescence in action – even in your drinking glass. Quinine – the bitter substance in tonic – is a chemical naturally derived from the bark of the cinchona tree that, when added to water, fluoresces blue under a black light (black lights emit long-wave ultraviolet, or UV, light). Fluorescent compounds like quinine absorb one wavelength of light and emit another, producing visible color. Sir John Herschel, the English polymath, first observed this phenomenon in 1845, and, in 1852, Irish physicist and mathematician Sir George Stokes deciphered the mechanism of fluorescence.
A few years later, English chemist Sir William Henry Perkin – just 18 years old at the time – attempted, on a bet, to synthesize quinine from coal tar. Though Perkin failed, he serendipitously succeeded in producing the first synthetic dye, a purplish substance called mauveine. When Perkin dipped silk into his creation, dying the fabric a reddish violet, he realized he was onto something (he would later industrialize the manufacture of synthetic dyes). With the door to synthetic dyes unlocked, thousands of dyes would be created in the decades to follow.
Some synthetic dyes were used as histological stains, coloring cellular structures for examination under a microscope. Then the worlds of synthetic dyes, biology, and fluorescence collided. In 1871, German chemist Adolf von Baeyer synthesized a fluorescent dye conveniently named fluorescein. And in 1882, German bacteriologist Paul Ehrlich used a variation of fluorescein to study fluid secretion in the eye. By the mid-twentieth century, synthetic fluorescent dyes were being used to label cellular structures, and by the year 2000, research teams were developing and patenting groups of fluorescent dyes that were better and brighter than their predecessors.
Fine-tuning fluorescent dyes involves measuring properties like brightness, which requires special instrumentation – a fluorometer. The history of the fluorometer takes us back to the quinine in your gin and tonic.
Quinine, and a synthetic dye called methylene blue, were both used as treatments for malaria. To discover how these antimalarial substances affected the malarial parasite and to determine the optimal dosages for treatment, doctors needed a way to measure fluorescence. In addition, the U.S. needed instruments to improve the manufacture of synthetic antimalarial drugs; during World War II, Imperial Japan controlled much of the cinchona tree plantations, leading to a shortage of quinine in the U.S. The invention of fluorometers resulted in more effective synthetic antimalarial compounds, an improved understanding of how they work, and better dosing.
Today, Luke works with the rhodamine class of dyes – initially synthesized in 1887 by the chemists Maurice Ceresole and Heinrich Caro while working with Ehrlich on derivatives of methylene blue. And the Lavis Lab has four fluorometers, now commonplace instruments, thanks to quinine. Building on the rhodamine dye scaffold, Luke’s group has discovered a set of novel azetidine-containing dyes, dubbed Janelia Fluor dyes, and continues to develop methods by which they and other researchers can create thousands of different colored fluorescent dyes for biological applications.