In Living Color: The Skinny on In Vivo Imaging Tools

If you start poking around on Addgene’s Fluorescent Protein Guide to In Vivo Imaging, you’ll pretty quickly notice the name Vladislav Verkhusha popping up again and again, and for good reason.

We all know scientists have used fluorescent proteins to observe what’s happening inside cells for at least a couple of decades. Green is the classic color, but fluorescent proteins are available in a variety of hues. While those tools are great for many applications, Verkhusha and his lab at Albert Einstein College of Medicine in New York recognized their limitations for peering right through living animals to see their organs – a liver or brain, say, or maybe a tumor. They wanted to find something better.

Transparency window

That’s exactly what they reported doing in a paper that appeared in Nature Biotechnology back in 2011 and then in another Nature Methods paper last year. The key to their bacterial phytochome-derived proteins, iRFP670, iRFP682, iRFP702, iRFP713 and iRFP720, is that they absorb and emit light in the near-infrared portion of the electromagnetic spectrum – the spectral region in which mammalian tissues are nearly transparent.

As Verkhusha explained it to me, the transparency window of mammalian tissues is set by the properties of hemoglobin and melanin pigments, which absorb the majority of light below 650 nanometers (nm), and the absorbance of water, which absorbs wavelengths above 900 nm or so. In other words, wavelengths between 650 nm and 900 nm will pass through animal and human tissues largely unimpeded.

The challenge then was to develop fluorescent proteins that would fall within that near-infrared range, and that’s exactly what iRFPs achieved. Those proteins allowed a signal-to-background ratio in mammalian tissues 20-fold greater than any fluorescent protein earlier described.

“Until our proteins, there were lots of fluorescent proteins made from jellyfishes and corals, but all of them fluoresce outside the transparency window of mammalian tissues,” Verkhusha said. “We developed near-infrared fluorescent proteins within this transparency window, so now, we could see deeper.”

Adding to the toolbox

In the last year, Verkhusha added two far-red light photoactivatable (PA) near-infrared fluorescent proteins (FPs), called PAiRFP1 and PAiRFP2, to the in vivo imaging toolbox, which increase their fluorescence upon illumination with far-red light. As the researchers described in Nature Communications last year, “The capability to control spectral properties of PA FPs with light of specific wavelength and intensity allows for optical labeling and tracking of proteins, organelles, and living cells in a spatiotemporal manner, which is not possible with conventional FPs. In addition, PA FPs can improve the achievable signal-to-background ratio, thus, allowing higher resolution in samples containing substantial autofluorescence background.”

Just last month in Scientific Reports, Verkhusha’s team reported another advance in the application of near-infrared fluorescent proteins, iRFP670 and iRFP720, as photoacoustic contrast agents for two-color imaging in animals. The approach relies on ultrasound waves as opposed to light to produce higher resolution images in vivo.

“One of the reasons we can’t localize one cell at a depth of one centimeter with fluorescence is because the light becomes very scattered,” Verkhusha explained. Ultrasound wavelengths, on the other hand, are larger than the size of a cell, so they scatter much less. As a result, ultrasound enables higher resolution images at depths up to eight millimeters – not single cells (yet) but small clusters of cells.

Looking ahead

What’s next, you might wonder? Verkhusha says they are always looking to improve upon existing genetically-encoded probes by making them brighter, more photostable, smaller, and less toxic to cells. They’d like to make near-infrared fluorescent proteins that operate within the 750 to 800 nm range, and his team is also looking in the direction of biosensors capable of revealing, for example, how muscles work inside a living animal.

For those making use of these probes, he says, when in doubt, read the methods sections of the relevant papers, and then read them again. Most of the time, the answers will be there.

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References:

Bright and stable near-infrared fluorescent protein for in vivo imaging. Filonov et al (Nat Biotechnol. 2011 Jul 17. doi: 10.1038/nbt.1918.)

Near-infrared fluorescent proteins for multicolor in vivo imaging. Shcherbakova and Verkhusha (Nat Methods. 2013 Aug; 10(8): 751-4. doi:10.1038/nmeth.2521)

Far-red light photoactivatable near-infrared fluorescent proteins engineered from a bacterial phytochrome. Piatkevich et al (Nat Commun. 2013 Jul 10;4:2153. doi: 10.1038/ncomms3153. PubMed)

Multicontrast photoacoustic in vivo imaging using near-infrared fluorescent proteins. Krumholz et al (Sci Rep. 2014 Feb 3; 4:3939. doi: 10.1038/srep03939.)

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