A Unique View of Disease

New outlook: A technique called Raman spectroscopy can image nanoparticles within the bodies of living mice, such as in the liver (shown here). The technique offers high sensitivity, low cost, and the ability to watch several molecules at once.
Credit: Sanjiv Sam Gambhir, Stanford University

Although scientists understand much about diseases like cancer on a molecular level, imaging diseases still relies largely on anatomy--the outline and shape of a tumor or a clot, for instance. Researchers have been working on ways to visualize molecular changes that take place inside the body, and a new method may offer some advantages over existing anatomical and molecular imaging technologies. In a study published online this week in the Proceedings of the National Academies of Sciences, a team of researchers at Stanford University used Raman spectroscopy, a technique common in chemical analysis, paired with specialized nanoparticles to noninvasively visualize organs and tumors in living mice. The technique could be useful for studying complex disease processes in animals, and, if found to be safe in humans, it could help clinicians view multiple molecular changes in certain cancers and other diseases.

Raman spectroscopy detects how objects scatter laser light, a phenomenon named after the Indian physicist Chandrasekhara Venkata Raman, who discovered the effect in the 1920s. Each type of molecule produces a unique Raman signature. There are several techniques that employ the Raman effect, but this study used SERS (surface enhanced Raman scattering), which relies on roughened surfaces of metal nanoparticles to greatly boost the Raman effect. To create Raman nanoparticles, scientists attach small dye molecules, which scatter light, to these molecular amplifiers. They can then affix molecules that allow them to target the particles to a location in the body, such as antibodies that bind to specific proteins in cells.

A study published last December in Nature Biotechnology, led by Shuming Nie at Emory University, used Raman nanoparticles to target and detect tumors in mice. In this study, the Stanford team used two kinds of Raman nanoparticles--a gold sphere and a carbon nanotube--as well as a specialized microscope to create images of the particles in living mice for the first time. The researchers imaged particles accumulating in the liver of the mouse, demonstrating that the technique could visualize structures within the body. They then used a tumor-specific nanoparticle to image tumors in mice.

The key advantage of this technique is that it allows for what imaging researchers call multiplexing: creating images of several different molecules at once. "One of the problems with imaging is, we tend to only be able to look at one or two things at a time," says Sanjiv Sam Gambhir, lead author of the study and codirector of the Molecular Imaging Program at Stanford. Multiplexing is important in complex diseases like cancer, in which several events occur within tumor cells, each of which could give information about the tumors' status and the likelihood that it will spread. As a first demonstration of multiplexing, Gambhir's team injected mice simultaneously with four kinds of Raman nanoparticles at different concentrations and showed that it is possible to locate the different particles and calculate their concentrations based on their Raman signal.

The most widely used molecular imaging technique in the lab is fluorescence. What makes Raman spectroscopy unique is that "you get a very sharp signal back, unlike [with] fluorescence, where you get a broad spectrum of energy," Gambhir says.

Claudio Vinegoni, an imaging specialist at the Center for Molecular Imaging Research at Harvard and at the Massachusetts General Hospital, who was not involved in the study, says that although scientists can use fluorescent molecules of different colors to see more than one molecule at a time, the ability to multiplex is limited because their signals quickly begin to overlap. In contrast, with Raman spectroscopy, "every molecule has its own Raman spectrum," Vinegoni says, so there is no possibility of the signals interfering. Because of their specificity, Raman nanoparticles can also be imaged at concentrations a thousand times lower than what can be detected using fluorescent quantum dots.

Although Raman spectroscopy could prove immediately useful in animal imaging, Gambhir ultimately hopes to bring it into the clinic. The best method for imaging biochemical events in humans is through PET imaging, in which a radioactive tracer injected into the body makes it possible to detect chemical activity. Gambhir's goal is to "develop the next generation of imaging technologies that wouldn't have to use radioactivity." In addition to its ability to image many things at once, Raman spectroscopy offers better sensitivity than PET and would be much less expensive.

One of the major shortcomings of this technique, as in all optical imaging methods, is the limited ability of light to penetrate deep into tissue. Although it can be used to visualize the internal organs of a mouse, Gambhir says that in humans, the technique would be more useful for visualizing tumors close to the surface of the skin, such as melanomas or even breast cancer. The technique could also be used in conjunction with endoscopes that probe inside the body. Gambhir's team is planning a clinical trial to test the use of Raman particles in conjunction with colonoscopies for detecting early-stage cancers. In this procedure, the nanoparticles could simply be sprayed onto the surface of the colon rather than injected into the body. But a key challenge for bringing this technique into the clinic will be determining the safety of nanoparticles as probes--studies that Gambhir's group is currently undertaking.

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