Intraoperative imaging of brain tumors is among the potential uses that could benefit from Raman imaging. Raman imaging has been explored for a range of uses; among the most exciting of these are applications in the biomedical arena. Here, especially in the past decade or so, considerable attention has been paid to the potential of the technique for diagnosis of disease. “One can imagine that Raman would provide something like a chemical histology map,” said Renato Zenobi, a researcher with ETH Zurich in Switzerland and an author of a recent review of Raman imaging, “where, instead of a stain, a real fingerprint pattern – for example, of a drug – can be used to detect the distribution in the tissue.” Several approaches to Raman imaging have been reported – including, for example, coherent anti-Stokes Raman spectroscopy (CARS), a multiphoton form of Raman spectroscopy that produces a coherent signal and, consequently, strengthens the emission, making it many orders of magnitude stronger than spontaneous Raman scattering. Researchers have demonstrated the potential of CARS for biomedical applications, including imaging of both ex vivo and in vivo biological samples, the latter including studies of atherosclerotic plaque deposits in animal models. Translation of the technology has proved more difficult, however. In the January 2013 issue of BioPhotonics, we noted that CARS had found itself at a crossroads: The technology had proved useful in the research lab, but developers were still wrestling with the question of how best to commercialize it. Early successes with lipid imaging had demonstrated the commercial potential of the technology, but efforts to develop it for other applications had been stymied both by cost and sensitivity. The SRS technique could offer an alternative to H&E staining for histopathology. Shown are SRS and H&E images of human glioblastoma multiforme xenografts. Thin sections of snap-frozen brain from an implanted human glioblastoma multiforme xenograft mouse model were imaged with both SRS and H&E microscopy (a). High-magnification individual fields of view demonstrating normal to minimally hypercellular cortex (75% tumor infiltration) (d). Courtesy of the Xie Group, Harvard University. This gave pause to developers of the technology. “We could one day reach a point,” said Yiwei “Kevin” Jia of Olympus America, who played a role in developing early CARS systems while at Harvard University in the early part of the past decade, “where we have to ask ourselves: Can we get the sensitivity to where it needs to be for this to become a general chemical imaging method, or do we just say ‘That’s it’? Might the best choice be to focus on the CARS method as [an] add-on modality of a multiphoton system?” But instead of simply giving up on CARS, researchers are working to push its sensitivity – or to evolve it into a new and improved technique. At the forefront of this effort is Sunney Xie, the Harvard researcher who pioneered the method. Xie and colleagues had been working to increase the sensitivity of CARS, but ultimately found that the challenges were too great because of the nonresonant (spectral) background. To address this, they developed a related technique based on stimulated Raman scattering, or SRS, reporting it in a 2008 Science paper. With this technique, the Raman signal is amplified when the difference in laser frequencies matches a particular molecular frequency – thus minimizing the spectral background responsible for the limited sensitivity of CARS. A new technique based on stimulated Raman spectroscopy helps to address the sensitivity issue with CARS. Shown are epi-SRS images of fresh normal brain slices. These include a full fresh 2-mm-thick coronal section (a) and structural features such as cortex (b), hippocampus (c), corpus callosum (d), choroid plexus (e), hypothalamic nuclei (f), habenular nucleus (g) and caudoputamen (h), all of which demonstrate the expected histologic architecture. Note that fresh-tissue images are free of the artifacts associated with fixation and freezing – techniques typically used for histologic imaging. Courtesy of the Xie Group, Harvard University. The researchers are now exploring a variety of possible applications. In a recent Science Translational Medicine paper, “Rapid, Label-Free Detection of Brain Tumors with Stimulated Raman Scattering Spectroscopy,” Xie and colleagues, including first author Minbiao Ji, described two of these. First, they showed that imaging ex vivo tissue slices with SRS could complement or perhaps even replace standard hematoxylin and eosin (H&E) staining, one of the most widely used approaches in the diagnosis of disease. SRS microscopy gave them much of the same information obtained simultaneously with H&E staining, but without the need for thin sectioning or tissue staining, which can lead to artifacts and other issues with the latter technique. They also showed that they could use SRS to delineate tumor tissue in a human glioblastoma multiforme xenograft mouse model, even identifying infiltrating tumor cells in areas that appeared normal to the eye. This is important because it is often difficult to distinguish between tumor and healthy tissue in the operating room. Two things can happen as a result: Surgeons can miss tumor tissue during resection, causing treatment failure and other poor outcomes. Just as significantly, they can mistakenly remove healthy tissue, leading to neurological deficits. The authors of the paper suggest that SRS microscopy could be developed for clinical application, to rapidly delineate areas of residual tumor on the surface of the cavity during surgery. The imaging depth is limited to 100 μm, they say, but the technique could be used iteratively to assess the edges of resection during the procedure. Challenges remain, of course, before SRS can be implemented in clinical settings. These include the fabrication of a handheld system for intraoperative use and obtaining the appropriate safety approvals. “The former will rely on the development of fiber lasers, fiber delivery and miniaturized scanner and objective lens,” Ji said. “The latter will be achieved with optimized laser parameters, including the pulse duration and repetition rate to minimize the total laser power without reducing signal levels.” The researchers are working to address all aspects of these challenges. For example, Ji said, they are developing fiber lasers and inventing electronics to cancel high-frequency noises. They are also working on a handheld device for SRS, using a MEMS mirror as the scanner.