Fluorescent dyes can help researchers visualize cell structure, but they can be difficult to use in more than a few.Photo: David Becker/SPL
When working with fluorescent dyes or proteins, researchers typically focus on two variables: the wavelength of light at which the molecules are stimulated to fluoresce, and the wavelengths at which they emit light, that is, their color. By balancing these properties, researchers can distinguish between about a half-dozen fluorescently labeled molecules in the same sample.
But there's more to fluorescence than just color, and scientists can now more easily exploit another property of fluorescent molecules to increase the number of proteins that can be imaged at once.
Led by Xin Zhang, a chemist at Westlake University in Hangzhou, China, the team designed more than two dozen fluorescent proteins that differ not only in color but also in how long they spend in an excited state—a property called fluorescence lifetime. The researchers call these molecules time-resolved fluorescent proteins, or tr-FPs. Their findings were published online last month in Cell1.
“This is brilliant work,” says Conor Evans, a physical chemist at Massachusetts General Hospital and Harvard Medical School in Boston. The team provided researchers with a “dialable palette” from which they could select both color and lifespan to get what they wanted, he said. “It's very powerful.”
Palette expansion
Suppose a scientist wants to map a cellular process associated with a particular protein. To determine where in a cell this process occurs, a researcher can use fluorescent dyes to highlight cellular landmarks—for example, blue for the nucleus, red for the cytoskeleton, and green for mitochondria. Viewing other proteins of interest against this background will require even more colors. However, because the visible spectrum is relatively narrow and fluorescent molecules emit light over a wide range of wavelengths, standard microscopes can only process a few colors at a time. After this, the emission spectra begin to merge, making it difficult to determine which signals come from which molecule.
The fluorescence lifetime makes it possible to expand this palette.
When a fluorescent molecule absorbs light, its electronic energy levels increase, entering what is called an excited state. The molecule hangs in this excited state for pico- or nanoseconds and then begins its luminous descent to the ground state, emitting excess energy in the form of photons. The time that a molecule spends in an excited state, that is, between the absorption and emission of photons, is called the lifetime.
To change the lifetime of existing fluorescent proteins, Zhang's team mutated certain amino acid residues to destabilize the region where the fluorescent signal is generated. The researchers then screened the resulting proteins to identify variants with different lifetimes but identical emission spectra as their wild-type counterparts. A total of 28 variants were produced, covering most of the visible spectrum.
Zhang's group then put this palette to work, pairing tr-FP with different target proteins and testing their behavior at different subcellular locations. They also examined how tr-FPs perform in a wide range of applications, including super-resolution microscopy, and showed that the technology can visualize nine target proteins in living cells using only three color channels.
Zhang notes that lifetime-based imaging is not a new concept, but he hopes tr-FP will help more researchers take advantage of the technique. “I think this is more of an addition to the large and powerful family of fluorescent proteins,” he says, rather than some kind of paradigm shift.
However, the researchers tested tr-FP in what they say is a new application: quantifying the relative concentrations of two proteins in a single living cell, something difficult to determine from fluorescence intensity alone.
