Technique Demonstrates Whole-body Fluorescent Scanning

Image courtesy of Vasilis Ntziachristos The arrow points to a lung tumor (red) that was reconstructed using optical fluorescence molecular tomography (FMT) and sits within a 3-D rendering of the skeleton and skin of a mouse based on computed tomography data.

Ever since green fluorescent protein (GFP) was cloned more than a decade ago, fluorescent proteins have become ubiquitous tools in the lab. By yoking these small beacons to other proteins or to gene promoters, scientists have literally illuminated countless processes in biology. Worldwide, organisms from yeast to pigs now glow with transgenic fluorescence.

But the use of such animal models is still limited by what we can see. A new imaging technique published in the Dec. 20 Proceedings of the National Academy of Sciences allows researchers to tomographically locate and quantify fluorescent proteins throughout the body in living mice. The method, developed by a team led by Vasilis Ntziachristos, HMS assistant professor of radiology at Massachusetts General Hospital, uses visible light to illuminate fluorescent proteins and to quantitatively reconstruct their location from inside tissues. Such a technology should enable researchers to track cells that express fluorescent proteins within the entire mouse body—for instance, to monitor the growth of tumors or the delivery of viral genes.

Adjusting the Light
Researchers have been limited in how deeply and how accurately they can image fluorescent proteins in vivo. Although microscopic imaging of cells expressing fluorescent proteins is common in vitro, applying this technology in vivo is difficult because tissue absorbs and scatters visible light, which most common proteins emit. In vivo fluorescent protein imaging is often based on confocal or multiphoton microscopy, but only to a depth of a few hundred microns. To get a look deeper into the animal, newer approaches use a tiny microscope inserted into an incision in the animal to watch biological events unfold. But on the level of an entire organism, the most common method is to simply take photographs of fluorescence using highly sensitive cameras.

This new method, rather than simply capturing fluorescence emitted back from within the animal, uses light in geometrical arrangements similar to those used in X-ray CT scans to provide information about the tissue it passes through. Beams of focused light are sent through an anesthetized animal at many different locations and angles. At the opposite end, a highly sensitive camera records both the transmitted light and the accompanying fluorescence. Rather than simply taking a picture of fluorescence, as many current methods do, this technique combines many images obtained at different light projections to calculate and reconstruct a three-dimensional image.

Photo by Graham Ramsay A whole-body imaging method developed by Vasilis Ntziachristos enables researchers to follow the activity of fluorescent proteins in live mice.

The behavior of light passing through tissues is much more complex than X-rays; visible light is strongly attenuated and bent. And matters are complicated further when light travels through organs and other structures, each of which has different properties. But recording the excitation light at the end of its journey “captures the optical heterogeneity in the tissue,” Ntziachristos said. Although the light’s path is complex, Ntziachristos’s team was able to devise a mathematical model to account for the tissue’s diverse optical properties and calculate the depth and degree of fluorescence. “You can correct your fluorescent signal and get the absolute amount of fluorescence,” he said. Without this adjustment, fluorescent signals would appear brighter when closer to the surface, while signals deeper or within denser tissues would appear weaker.

The Animal Evidence
The team demonstrated the method in three mouse models. In the first, they imaged tumors implanted superficially, measuring reflected light and then transmitting light through the animal. The experiment showed that while reflected light gave a brighter signal, it was marred by the skin’s own strong autofluorescence. In a second experiment, the team used their tomography method to image deeper tumors of the lung, showing that the technology can go beyond the surface. A third experiment imaged tumors that expressed yellow fluorescent protein that were infected with a virus expressing green fluorescent protein, showing that the technique could be used to capture multiple fluorescent signals.

Ralph Weissleder, HMS professor of radiology at MGH and a co-author of this paper, said that the study “shows for the first time that you can actually quantify the number of green fluorescent proteins” in a living animal. Though it provides a broad view of the activity of fluorescent proteins in the body, the method lacks the resolution of microscopic imaging. The new method can probe deeper than microscopy, but it is still limited to a depth of a little over a centimeter—perfect for mice, but too shallow for larger animals. Ntziachristos said that the development of new fluorescent proteins in the far-red, which are absorbed less strongly, will make it possible to achieve better sensitivity and tissue penetration. The team is currently working to improve the method’s ability to accommodate multiple proteins from a larger pool of available fluorescent molecules.

Jason Gaglia, HMS clinical fellow in medicine at Beth Israel Deaconess Medical Center, is currently working with the team to apply this method to image islet cells in the pancreas. He has also successfully used the system in pilot experiments. With protein tomography, he said, “You get spatial information. You can quantify how much tissue is fluorescent.”

Weissleder explained that fluorescent tomography “doesn’t replace anything; it’s really additive to existing technology.” He and Ntziachristos envision a whole-body imager as one of the tools of biomedical labs along with in vivo microscopy. Such a machine would be especially useful for tracking proteins over time—for instance, by tagging tumors and watching how their volume changes or monitoring the distribution of tagged cells in an animal’s body. Often, studies like these require sacrificing animals at each time point and piecing together the course of events from this information. Using whole-body imaging, researchers could follow changes in an individual over time, using fewer animals.

—Courtney Humphries

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