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By pushing fluorescent proteins further into the red, bioengineers are expanding the palette and penetration depth of biological imaging. By Amber Dance
Green fluorescent protein is one of the most popular items in the microscopist's toolbox. It is a Nobel-prizewinning innovation that brilliantly lights up molecules of interest across a diverse range of biological fields, laboratories and techniques. But it does not work for physical chemist Julie Biteen.
Biteen studies gut bacterial communities at the University of Michigan in Ann Arbor, and was eager to use fluorescent proteins to identify individual species in complex mixtures. But gut bacteria don't like oxygen - something green fluorescent protein (GFP) absolutely requires. No oxygen, no fluorescence.
So, she turned to a label that can do without oxygen. A relatively new addition to the fluorescent-protein palette, IFP2.0 fluoresces mainly in the near-infrared - a portion of the electromagnetic spectrum that is barely visible to the human eye but readily apparent to microscope cameras1. "We're really excited," Biteen says. "We could see single cells and identify them."
Imaging at the red end of the spectrum offers other advantages, too: lower background fluorescence, reduced toxicity and deeper tissue penetration. "All other factors being the same, redder is better," says Robert Campbell, a protein engineer who spends half his time at the University of Tokyo and the other half at the University of Alberta in Edmonton, Canada. It also provides a way to add another hue, or two, to experiments. "The more channels we can pack into an experiment, without significant bleed-through, the more interactions we can study," says Talley Lambert, a microscopist at Harvard Medical School in Boston, Massachusetts.
Reddish fluorescent proteins have existed for decades, but they are still generally no match for GFP in terms of both brightness and hue. Even the 'red' fluorescent protein RFP is closer to orange. Scientists are making headway in developing fluorescent proteins that are truly red - often called 'far red' to distinguish them from earlier attempts. Infrared offers similar advantages. Development is still in its infancy, but advances in bioprospecting, protein engineering and synthetic chemistry are helping to improve the labels. Most are available, in gene form, on the plasmid repository Addgene.
There's clearly a need. Scientists are desperate for tags...