But Raman has its limitations — particularly, when it comes to the very low number of photons produced. In the 1970s, researchers discovered that if Raman-active molecules were coupled to certain metallic nanostructures, dramatic signal amplification could result — in some cases up to 15 orders of magnitude. This effect was termed “surface-enhanced Raman spectroscopy” (SERS). Still, another 35 years would elapse before two research labs, working independently, would combine these discoveries and make Raman imaging a viable choice for biological labs.Picture Perfect
The first paper, published in January 2008, came from the lab of Shuming Nie, the Wallace H. Coulter Distinguished Faculty Chair in Biomedical Engineering at Emory University (1). Led by research assistant professor Ximei Qian, a physical chemist-turned-biomedical engineer, Nie's team prepared SERS-active particles by coating 60-nm colloidal gold with Raman-active compounds, such as malachite green or diethylthiatricarbocyanine iodide, and then with a polyethylene glycol shell spiked with a molecule that recognizes EGFR on the surface of cancer cells. When the team injected those particles into the tail veins of nude mice harboring a human head-and-neck tumor expressing EGFR, the particles concentrated in the tumor over 4 to 6 hours, an effect the research team observed by illuminating with a 785-nm NIR laser coupled to a Raman spectrometer.
The second paper, published in April 2008, came from Gambhir's lab. (2) An MD/PhD from UCLA who did his thesis work in applied mathematics, Gambhir helped establish the field of molecular imaging, the union of traditional imaging modalities with the molecular specificity of antibodies, receptors, and pro-substrates. “Imaging tends to have a lot of mathematics because of image reconstruction and modeling of how imaging agents behave in the body,” he says.
In the mid-2000s, Gambhir was testing nanoparticulate quantum dots for their use in vivo. Frustrated by their limitations, notably in terms of cytotoxicity and multiplexing, he began looking for other potential imaging strategies. “By going into the literature we said, ‘oh, there may be a way to make a parallel technology work that doesn't rely on quantum dots but still uses nanoparticles. And, because they may not be toxic, maybe we should try them.”
Whereas quantum dots are made of toxic semiconductor materials like cadmium and selenium, SERS-active compounds are made mostly of silver, gold, and platinum. Traditional SERS substrates are planar surfaces. But like Nie, Gambhir's team opted for gold nanoparticles – specifically, an off-the-shelf formulation comprising a gold core, Raman reporter, and silica shell.
The team imaged whole mice by raster scanning their NIR laser over the animal's body. They used a modified Renishaw InVia Raman microscope – basically a confocal microscope coupled to a Raman spectrometer, which was further customized for small animal imaging with new optics and a computer-controlled stage. A complete scan took 20–25 minutes, Gambhir says, compared to 1 minute using fluorescence and bioluminescence imagers. Each point was imaged for 3 seconds, and dedicated software converted the spectral peaks into colored pixels. The resulting image is like a red-green-blue JPEG, with each color channel mapping to a single Raman reporter.
Gambhir also tested a non-metallic option, single-walled carbon nanotubes (SWNT) used either as-is or coupled to a targeting peptide. Carbon nanotubes, as Gambhir notes in his article, “show an intense Raman peak produced by strong electron–phonon coupling that causes efficient excitation of tangential vibration in the nanotubes quasi one-dimensional structure upon light exposure.” Simply put, the unique electronic structure of a SWNT allows it to act as both Raman reporter and SERS amplifier. A 2010 study led by Hongjie Dai at Stanford University showed that SWNTs bearing different C13/C12 ratios produce distinct Raman signals, meaning the nanotubes may be multiplexed – an important consideration in biological imaging.
In its earlier 2008 study, Gambhir's team demonstrated that Raman imaging with nanoparticle probes could also support multiplexing. They injected four reporters at separate sites in the mouse body, plus a mixture of the four at a fifth site. The resulting image shows the injection sites as a series of low-res pixels in varying shades of primary colors — green, red, yellow, and blue, while the fifth injection site appears purple, thanks to an abundance of red and blue particles.