Named for the Indian physicist and Nobel Laureate C.V. Raman, Raman imaging has long-standing applications in areas such as materials science, chemistry and even art history. Here, we look at biomedical applications – in particular, at Raman imaging – focusing on recent developments that could lead to advances in tissue imaging. Recent years have seen an increase in the use of gold nanorods (GNRs) as contrast agents for in vivo optical imaging, with applications including near-infrared transmission imaging and photoacoustic tomography as well as surface-enhanced Raman scattering (SERS) imaging. With deep tissue imaging, however, nanorods are still limited, especially by signal attenuation and low contrast. To overcome these problems, investigators with the Royal Institute of Technology in Stockholm, Sweden, and Zheijiang University in Hangzhou, China, have developed an optically multifunctionalized nanoplatform based on gold nanorods. As described in a recent issue of Biomaterials (32, 2011, pp. 1601-1610), they functionalized the particles with near-infrared fluorescence and surface-enhanced Raman scattering. With this strategy, “Raman scattering of reporter dyes was greatly enhanced due to the LSPR (localized surface plasmon resonance) effect of the GNRs. [At the same time], the dyes’ NIR fluorescence remained sufficiently bright when the GNRs were surface-modified with PEG [polyethylene glycol] and their LSPR band was appropriately adjusted,” said Jun Qian, first author of the paper. Researchers have reported gold nanorods (GNRs) functionalized with both near-infrared fluorescence and surface-enhanced Raman scattering. The nanorods could contribute to a range of applications, including photodynamic therapy. Courtesy of Jun Qian and Sailing He, Zheijiang University. A broad range of applications could benefit from this work. In the Biomaterials paper, the researchers demonstrated use of the multifunctionalized nanorods for sentinel lymph node mapping and tumor targeting in mice. Qian also noted that the nanorods have potential for photodynamic therapy. At Stanford University in California, Sanjiv S. Gambhir’s group is developing nanoparticle-based imaging of colon cancer via Raman spectroscopy. In the past several years, the researchers have demonstrated picomolar sensitivity with the nanoparticles and have shown that they can multiplex and separate up to five Raman signatures. A team at Stanford University is developing Raman nanoparticles for molecular imaging of EGFR, a cell-surface biomarker associated with colon cancer. The researchers use 60-nm gold nanoparticles encased in a silica shell for Raman imaging. The Raman signal originates from a dye immobilized on the surface of the nanoparticle (red). To link the particle to the target, stabilizing and targeting ligands are added (green). Courtesy of Jesse V. Jokerst, Stanford University. Now, in a Small paper published on the journal’s website on Feb. 8, 2011, the researchers report steps toward clinical application – namely, the construction of Raman nanoparticles for molecular imaging of the epidermal growth factor receptor (EGFR), a cell-surface biomarker commonly found in colon and other cancers. With this work, the researchers hope to enhance the efficacy of colonoscopy in detecting cancers. Colonoscopy is a well established procedure that has been shown to reduce mortality by 30 percent, but it does not provide molecular insight into the nature of the lesions it finds. Nor can it detect so-called “flat” lesions – those that do not protrude from the colon wall. A technique that adds molecular imaging capabilities to the existing benefits of colonoscopy, the investigators wrote, could lead to increased early detection rates and improved patient outcomes. The Stanford investigators are working toward clinical implementation of the technique. Here, clinicians would administer the nanoparticles and, with specially designed hardware integrated with existing colonoscopy instrumentation, would identify tumor areas based on the Raman signal they give off. Courtesy of Cristina Zavaleta, Stanford University. To this end, they developed gold core silica-clad nanoparticles functionalized with an affibody ligand targeted to EGFR, offering both high affinity and long-term stability. Nanoparticles will be administered via an enema during the routine bowel preparation that a patient undergoes prior to colonoscopy. Diseased tissue expressing high levels of EGFR will have more Raman signal and will guide the endoscopist in removal of the lesion. Results with the particles with animal models of human disease were promising. In experiments described in the Small paper, the signal was almost 35-fold higher in EGFR-positive than in EGFR-negative tumors. At the same time, expression in the surrounding healthy tissue was sevenfold lower than in the EGFR-positive tumors. The researchers continue to develop the particles for molecular imaging of EGFR. Here, one of the challenges is achieving the proper distribution in the body. “We’re getting the particles from a security company that uses them for counterfeiting applications,” said Jesse V. Jokerst, first author of the paper. “They want bright and stable; they’re not superconcerned about the biodistribution properties.” To improve these properties, he is modifying the shape of the particles – moving from a large spherical shape to a smaller rodlike design. This class of smaller particles will leave the vasculature to label cancer cells after intravenous injection, unlike the current particle generation, which is limited to the colon. “Currently, the only way to measure multiple tumor biomarkers simultaneously is via biopsy,” Jokerst said. “Raman imaging offers a way to do this in real time in vivo, which has significant implications in both early detection and monitoring cancer’s response to therapy.” As a further step toward clinical application of the technique, Stanford researchers including Cristina Zavaleta and Ellis Garai are working to integrate it with existing colonoscopy instrumentation. With their Raman-based strategy, Zavaleta said, the nanoparticles would serve as “tumor-targeting beacons.” The clinician would administer them into the body and then, with the endoscope, find tumor areas based on the Raman signal they give off. “This is very different from what has predominately been done thus far, with utilizing Raman spectroscopy to look at intrinsic signal changes due to differences in the chemical compositions of the tissues themselves (cancer versus normal tissue),” she said. The researchers have faced challenges in developing the technology. For example, they had to ensure that the Raman endoscope could be sent through the accessory channel of a conventional endoscope without damaging the instrument. Most endoscopes have an angular bend at the opening of the accessory channel, so, to avoid scratching or other damage, they had to make the instrument less rigid at the head. They also are working to address inconsistencies with the optics noted during the Small study – designing the scope to be less vulnerable to varying working distances, for example, to provide a more reliable and representative signal. Others working to develop hardware for Raman techniques include a team of researchers from Vanderbilt University, from Vanderbilt University Medical Center, and from the Tennessee Valley Healthcare System, all in Nashville, Tenn.; and from the MIRA Institute for Biomedical Technology and Technical Medicine in Enschede, and from the University of Amsterdam, both in the Netherlands. This group has been developing a combined Raman spectroscopy (RS)-OCT instrument for optical analysis of tissues. The team previously developed the combined instrument and demonstrated the complementary benefits of the two techniques for characterization of tissues. The device was built on separate Raman spectroscopy and time-domain OCT hardware backbones and integrated with common sampling optics. “The system was effective,” said researcher Chetan A. Patil of Vanderbilt University, “but it required extensive instrumentation and utilized a detection scheme for OCT that could be considered a generation behind the state of the art.” The researchers therefore explored the feasibility of developing a combined instrument using a single spectrometer for detection of both the Raman spectroscopy and OCT signals, describing their findings in the January 2011 issue of the Journal of Biomedical Optics. A combined Raman spectroscopy-optical coherence tomography (OCT) system could help to advance tissue characterization. Shown is a schematic of the system. PC = polarization control paddles, ND = neutral density filter; WC = water-filled cuvette; TM = translatable mirror; LP = long-pass filter; DM = dichroic mirror; BP = bandpass filter; SF = spatial filter; X-Y = X-Y galvanometer pair; MOS = microelectromechanical systems optical switch; and NI-DAQ = National Instruments multifunction data acquisition. Courtesy of the Journal of Biomedical Optics. “It’s well known that spectrometer-based OCT detection schemes can offer potential advantages in imaging speed and sensitivity over time-domain detection schemes,” said Patil, first author of the paper. “The design we reported enabled a reduction in the overall hardware requirements for RS-OCT as well as provided a proof-of-principle demonstration of an RS-SD [spectral domain] OCT instrument.” Because of the enormous dynamic range of photons the two modalities seek to detect and the different acquisition times needed, “we were pulling our detector in two different directions, and pushing the flexibility of its performance potential to near its limit,” he added. To address this, the researchers used a detector that prioritizes Raman spectroscopy performance. The detector worked well with ex vivo and in vitro samples, but the compromise affected the imaging speed and sensitivity of OCT and resulted in limitations when investigating in vivo samples. The researchers continue to look for different configurations with which to optimize the performance of the system. At the same time, they are developing applications for the first-generation instrument, using separate detectors for Raman spectroscopy and OCT. These include skin cancer screening and diagnosis, and other applications where the combination of Raman spectroscopy and OCT may be beneficial. Increased multiplexing capabilities In Germany, researchers at the University of Osnabrück, the University of Würzburg and Wilhelm Conrad Röntgen Research Center, also in Würzburg, also are developing functionalized metal nanoparticles for use with SERS microscopy. Researchers have described the design and synthesis of Raman reporter molecules for tissue imaging by immuno-SERS microscopy. Shown is the synthesis of SERS-labeled antibodies, beginning with Au/Ag nanoshells. Courtesy of Max Schütz, University of Osnabrück. Previously, they used commercially available aryl thiols as Raman reporter molecules with Raman bands at ~1100 and ~1600 cm-1. Wanting to increase the multiplexing capacity and to obtain higher SERS signals, they sought new reporter molecules with Raman bands above 1600 cm-1 and high Raman cross sections without losing sight of the other requirements, such as the thiol group. In a Journal of Biophotonics paper published online on Feb. 7, 2011, the researchers reported Raman reporter molecules designed to meet their criteria: unique Raman bands; high Raman cross sections and, therefore, high Raman intensities; a thiol group for binding to the surface of Au/Ag nanoshells; and an ability to achieve a stable self-assembled monolayer. Any number of applications could benefit from use of these reporters, said Max Schütz of the University of Osnabrück, particularly applications such as SERS microscopy and SERS immunoassays, which could take advantage of the increased multiplexing capacity.