Fluorescence microscopy cannot easily reveal internal details of a living animal, in part because fluorescence cannot easily penetrate through skin. Although near-infrared emission improves the penetration of fluorescence through skin, the lack of available fluorophores without substantial overlap in that region makes simultaneous imaging of multiple molecular targets a difficult endeavor. Furthermore, autofluorescence signals from skin often obfuscate the fluorescence signal.In contrast, multiple labels can be resolved easily using Raman spectroscopy, and the Raman signal can be separated from autofluorescence without difficulty because the signal comes from a type of inelastic photon scattering and not from fluorescence emission. Additionally, the Raman signal lasts indefinitely, whereas fluorophores eventually photobleach. Therefore, Raman spectroscopy enables longer-duration experiments.Although Raman scattering usually is weak, strong Raman signals can be elicited from substances such as carbon nanotubes and gold nanoparticles that exhibit the surface-enhanced Raman scattering (SERS) effect. These substances can be used as labels for biological molecules.Because Raman spectroscopy has several advantages over fluorescence microscopy for imaging living animals, Dr. Sanjiv Sam Gambhir and members of his laboratory at Stanford University in California have developed a technique based on Raman spectroscopy that enables the imaging of molecular targets deep within living mice using nanoparticle labels. Gambhir envisions this technique as a research tool that will accelerate studies of disease progression and response to treatment, including drug discovery. He emphasizes that, although no single imaging modality has all the traits desirable for in vivo whole-animal imaging, his Raman technique delivers high sensitivity and the ability to detect multiple molecular targets simultaneously. These are important advantages for applications such as cancer research.The nanoparticles used included single-walled carbon nanotubes and Nanoplex Biotags from Oxonica Inc. of Mountain View, Calif., gold nanoparticles coated with silica on top of a molecular layer active for SERS. The chemical composition of the molecular layer varied depending on the tag that was used.The researchers based their setup on a Renishaw InVia Raman microscope equipped with a 785-nm excitation laser (Figure 1). They made several modifications to the microscope to optimize it for imaging a live mouse, such as replacing the high-NA objective on the microscope with a 12× lens that enabled a wider field of view. The wide-field lens also resulted in a defocused beam with a spot size of 20 × 200 μm, which prevented the laser from burning the skin of the animal.Figure 1. Researchers developed this system for Raman imaging entire living mice using nanoparticle labels. Images reprinted with permission of PNAS.To enable raster-scanning of the mouse, a computer-controlled X-Y translation stage moved the animal. The investigators attached the mouse to an anesthesia unit to prevent it from moving, which would blur the images, and they put a heated bed on the stage to keep the mouse at a constant temperature during anesthesia. Because the Raman imaging system currently raster-scans the mouse slowly line by line, they plan to integrate wide-field imaging into the system to speed up the process in future work and to perform tomography. “[Tomography] requires multiple views from multiple angles and a reconstruction of the image on a slice-by-slice basis,” Gambhir said. Tumor targetingThe researchers detailed the current experiments in the April 15, 2008, issue of PNAS. In one experiment, they injected solutions of SERS nanoparticles, each with unique Raman peaks, at various concentrations under the skin of the mice. So that they could image multiple SERS labels simultaneously, they opened the monochromator slit 100 μm wide for a spectral resolution of 10 cm21. They demonstrated that they could monitor at least four different SERS tags under the skin at the same time. Gambhir said that they potentially could distinguish as many as 10 to 30 labels at once.In the main experiments, the scientists performed Raman imaging of entire living mice. They injected either the carbon nanotubes or SERS nanoparticles into the tail vein of the mice and watched as the nanoparticles migrated to the liver. The carbon nanotubes also enabled the researchers to monitor tumors in the living mice because the surface of the nanotubes contained peptides for targeting alpha-v-beta-3 integrin, which is heavily produced by tumor blood vessels (Figure 2). The researchers monitored glioblastomas, one of the most aggressive and deadly tumors. Figure 2. The researchers used the system for several purposes, including imaging tumors labeled with carbon nanotubes as shown here. The nanotubes carried arginine-glycine-aspartate (RGD) peptides targeting alpha-v-beta-3 integrin, a protein heavily produced by tumor blood vessels. The peptide-labeled nanotubes targeted tumors much more effectively than plain nanotubes. (SWNT = single-walled carbon nanotubes).They also used this study as an opportunity to gather data on the uptake of both nanoparticles by the liver because macrophages of the reticuloendothelial system, which includes the liver, usually uptake and destroy nanoparticles that otherwise would be viable for imaging or drug delivery. Gambhir said that these data will enable them to design nanoparticles with less accumulation in the reticuloendothelial system.In the April 2008 issue of Nature Nanotechnology, the Gambhir laboratory also reported that the same carbon nanotubes used in the PNAS study are nontoxic in mice followed over four months. The FDA requires significant toxicity data before approval for use in humans. The investigators have planned a clinical trial to use Raman spectroscopy to detect colorectal cancer in humans using endoscopes. Gambhir said that this strategy could be used to detect esophageal, cervical and other cancers as well.