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Near-infrared Fluorescent Proteins for Deeper Tissue Imaging

Kelly Rae Chi

Newly developed near-infrared fluorescent proteins have the potential to allow deeper imaging in intact, living tissue. How do they work? Find out...

Phytochrome proteins from soil bacteria can sense red and far-red light using naturally occurring biliverdin as the chromophore. Because biliverdin is also present throughout the bodies of mammals, bacterial phytochromes have the potential to enable imaging of living mammalian cells, tissues, and even whole animals.

Now, new near-infrared fluorescent proteins made from bacterial phytochromes have been developed. These proteins, which facilitate imaging deeper into living tissues and small animals, were recently described in Nature Methods, Nature Communications, and Chemistry & Biology (1,2,3).

Early efforts to engineer bacterial phytochromes for this purpose damaged them, preventing binding of endogenous biliverdin, said Vladislav Verkhusha, senior author of all three studies, at Albert Einstein College of Medicine in New York. In contrast, “we preserved the high affinity, high specificity of bacterial wild type phytochromes in the resultant fluorescent proteins,” he added.

In a study published in Nature Methods, Verkhusha’s group developed four spectrally distinct near infrared proteins: iRFP670, iRFP682, iRFP702, and iRFP720. When expressed in the tumors of a mouse xenograft breast cancer model, these new proteins were visible using whole-body epifluorescence as early as one week after the cancer cell injection.

When the researchers implanted two tumors expressing either iRFP670 or iRFP720 close together in a single mouse, they were then able to distinguish the tumors in the whole animal.

Absorbance by hemoglobin in the blood and melanin in the skin have long been limiting factors for imaging deep into living tissue. But light of certain wavelengths—from about 650 nm to 900 nm—can penetrate deeper. Depending on the tissue type, Verkhusha and colleagues can currently image animal tissues up to 20 mm deep using their new bacterial phytochrome-derived probes.

Two additional near-infrared proteins, described 10 July in Nature Communications as PAiRFP1 and PAiRFP2, are photoactivatable. To turn on the proteins, researchers used far-red light of low power to illuminate the skin outside of an implanted tumor expressing the proteins, or they injected the light into the backside of the tumor using a needle and light guide. The area that they illuminated was several cubic millimeters.

One advantage of photoactivatable iRFPs is that researchers can subtract non-photoactivated images from those that are photoactivated, which removes a majority of the noise caused by autofluorescence, Verkhusha said.

In the third study, published online by Chemistry & Biology, Verkhusha’s team developed a split probe using a bacterial phytochrome-derived near-infrared fluorescent protein that can report on protein-protein interaction. When the two halves of the probe come together, it fluoresces.

In that study, the researchers linked half of the probe to the protein FRB and the other half to FKBP, a protein that complexes with FRB only in the presence of rapamycin. These newly joined molecules were expressed both in vitro and in vivo in a mouse breast tumor. When the team injected rapamycin into living mice, fluorescence of the tumor increased by up to 23-fold at 36 hours after injection.

Although these proteins have the advantage of being less cytotoxic than commercially available (GFP-like) red fluorescent proteins, their molecular brightness is still fairly low. This makes them less optimal for standard microscopy applications using cultured mammalian cells, for example, said Verkhusha.

Verkhusha and his group have provided all of their new fluorescent proteins, including the split probe, to the research community through Addgene.


1. Shcherbakova D.M. and Verkhusha V.V. Near-infrared fluorescent proteins for multicolor in vivo imaging. Nature Methods 2013, 10: in press. doi:10.1038/nmeth.2521

2. Piatkevich, K.D., Subach F.V. and Verkhusha V.V. Far-red light photoactivatable near-infrared fluorescent proteins engineered from a bacterial phytochrome. Nature Communications 2013, 4: 2153. doi:10.1038/ncomms3153

3. Filonov G. S. and Verkhusha V.V. A near-infrared BiFC reporter for in vivo imaging of protein-protein interactions. Chemistry & Biology 2013, 20: in press. doi: 10.1016/j.chembiol.2013.06.009

Keywords:  Fluorescent protein