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In vivo imaging: Raman-style
 
Jeffrey M. Perkel, Ph.D.
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The team next injected these particles into tail veins of live mice. When imaged two hours later, Raman-active particles could be located without knowing where they were a priori (they had accumulated in the liver). When peptide-conjugated SWNTs were injected into mice bearing a human tumor, they homed to the tumor over 24 hours, whereas non-peptide-conjugated nanotubes did not. Critically, these particles could be detected even when there were very few of them to find. In its 2008 paper, Gambhir's team could see as few as 600 particles. Today, he can measure at close to the 10-attomolar range. The technique is “exquisitely sensitive,” he says.

Raman on the ward

An obvious application for in vivo Raman imaging would be in oncology, where Raman's (or more properly, SERS's) “exquisite” sensitivity could help a physician ensure that a tumor is truly gone.

In 2011, Nie and Qian used their SERS nanoparticles and a handheld Raman probe to test blood samples for the presence of circulating tumor cells (CTC), those rare tumorigenic seeds that can lead to metastases wherever they land (3). The technique was sensitive enough to detect as few as one CTC per milliliter of whole blood.

For his part, Gambhir in 2012 described a tri-functional nanoparticle useful in magnetic resonance imaging, photoacoustic imaging, and SERS (4). The new particle, called an MPR (MRI-photoacoustic imaging-Raman) nanoparticle, is a 60-nm gold structure coated with a thin layer of trans-1,2-bis(4-pyridyl)-ethylene (a Raman reporter) and then again with a silica shell, and finally with gadolinium, making it MRI-responsive. When the team injected these particles into the tail veins of mice harboring a human glioblastoma, the particles accumulated in the tumor via the so-called “enhanced permeability and retention” (EPR) effect, a disruption of the blood-brain barrier, and could be detected using any of the three modalities. This was the tumor that was resected from that mouse brain earlier in the story, and Raman played a critical role in its detection and removal.

“The Raman part was very important because it let us see individual tumor cells that would have otherwise been totally missed,” Gambhir says — a development that, if replicated in humans, could dramatically improve long-term prognoses.

Of course, to achieve such benefits, physicians and researchers need access to user-friendly Raman equipment. “Technology based on Raman scattering is fairly specialized, so it requires more instrumentation than fluorescence,” says Nie. Indeed, Nie notes that relatively few groups have the capacity to use Raman for in vivo imaging, his and Gambhir's being among the most prominent.

“It's a concept foreign to the biological sciences community,” Nie says. But the development of “cheap devices” and off-the-shelf SERS particles could help, he says.

Off-the-shelf SERS particles do exist; Gambhir uses them in his studies. But new designs are also in development. Washington University mechanical engineer Srikanth Singamaneni recently described a new SERS particle design dubbed BRIGHT — “bilayered Raman-intense gold nanostructures with hidden tags.” (5) Unlike the core-shell structures Nie and Gambhir have used, BRIGHTs have a gold core, a thin Raman label shell, and then a gold coating, resulting in particles that produce some 20-fold brighter signals than traditional particles according to Singamaneni — at least in vitro.

On the hardware front, Nie's group has developed a dedicated handheld Raman spectroscope called a SpectroPen, and tested it in mice. The device, being commercialized through an Emory spinoff company called SpectroPath (for which Nie is Chief Scientific Consultant), is a pen-sized gadget that will allow surgeons to identify tumor margins following SERS particle injection based on a beeping sound, similar to a contractor locating wall studs with a stud-finder.

Gambhir's lab is developing a fiber-optic endoscopy add-on that enables imaging of SERS particles in, for instance, the colon wall of human patients. According to Gambhir, the tool allows multiplexed detection of up to 10 different Raman reporters over a wide working distance, which is critical when dealing with the uneven contours of living tissue. The lab, in collaboration with GE, is also working on a high-speed, dedicated Raman imager, called SARI (small animal Raman imaging).

The result of all this development work could be in vivo imaging orders of magnitude better, in terms of both sensitivity and multiplexing, than currently achievable with optical modalities. That's not to say fluorescence and bioluminescence are going anywhere. “The advantage of fluorescence is you can get fluorescence from small molecules; you don't have to inject gold nanoparticles,” says Gambhir. “Also, there are already instruments available for it.” Plus, Raman can really only be used for relatively shallow imaging, up to 1-cm deep. “You could go into a mouse skull, but not into a rabbit or human skull,” he says, meaning human brain imaging, for instance, would have to be carried out in the operating theater.

In the end, says Gambhir, there's room enough for multiple modalities in clinical imaging. “There are advantages and disadvantages for fluorescence and bioluminescence and Raman. There's not a single clear winner.” Well, other than patients, of course.

References
1.) Qian, X.. 2008. In vivo tumor targeting and spectroscopic detection with surface-enhanced Raman nanoparticle tags. Nat. Biotechnol. 26:83-90.

2.) Keren, S.. 2008. Noninvasive molecular imaging of small living subjects using Raman spectroscopy. Proc. Natl. Acad. Sci. USA 105:5844-5849.

3.) Wang, X.. 2011. Detection of circulating tumor cells in human peripheral blood using surface-enhanced Raman scattering nanoparticles. Cancer Res. 71:1526-1532.

4.) Kircher, M.F.. 2012. A brain tumor molecular imaging strategy using a new triple-modality MRI-photoacoustic-Raman nanoparticle. Nat. Med. 18:829-834.

5.) Gandra, N., and S. Singamaneni. 2013. Bilayered Raman-intense gold nanostructures with hidden tags (BRIGHTs) for high-resolution bioimaging. Adv Mater. 20 25:1022-7.

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