17 Apr 2008
Raman spectroscopy offers an alternative to fluorescence techniques for molecular imaging of living subjects.
Molecular imaging of living subjects is a hot topic, with fluorescence techniques in particular being developed for in vivo small-animal studies. But there's another option on the horizon: Raman spectroscopy. Raman offers some big advantages over fluorescence for biomedical applications – including high multiplexing capability and lack of background autofluorescence signal. Now, researchers at Stanford University School of Medicine (Stanford, CA, US) have used Raman spectroscopy to visualize nanoparticles deep within living animals (PNAS doi: 10.1073/pnas.0710575105).
Raman spectroscopy works by detecting inelastic light scattering, in which a photon striking a target scatters with a wavelength shift characteristic of the target material. While it's an inherently weak effect – just one in around 107 photons striking a target will scatter inelastically and contribute to the Raman signal – enhancement by proximity to a rough metal surface (surface-enhanced Raman spectroscopy, or SERS) can ramp intensities to strengths comparable to fluorescence signals.
The Stanford researchers used SERS-active nanoparticles – comprising a gold core, a Raman-active molecular layer and a silica coating – to perform non-invasive imaging of living mice. Weak solutions of the nanoparticles were injected into the mice. Upon illumination from an external near-infrared (785 nm) laser, Raman signals from the nanoparticles were detected from both superficial and deep tissues.
"The light penetrates through the skin without damaging it and interacts with the Raman nanoparticles, which produce a signal that's detected by an imaging system placed outside of the mouse," explained team leader Sanjiv Sam Gambhir, professor of radiology and director of the Molecular Imaging Program at Stanford (MIPS). "It is an intact mouse, no surgery or exposure of internal organs is required."
The technique exhibits sensitivity on a picomolar level – making it 1000 times more sensitive than quantum-dot-based fluorescence imaging. "This means, for instance, that if we injected a 20 microlitre volume under the skin of a mouse, then we need about 100 million SERS nanoparticles in that volume (8.125 pM) to see them with our Raman microscope," explained first author Cristina Zavaleta, NCI postdoctoral fellow at MIPS. "On the other hand, it takes 100 billion quantum dots in that same volume for detection with current optical imaging devices."All in one
Another of Raman's advantages over fluorescence spectroscopy is that it can differentiate the unique spectral fingerprints of many different molecules within one target area. To test these multiplexing capabilities, the Stanford researchers injected mice with four distinct types of SERS nanoparticle (each with a unique Raman spectrum) at separate sites, plus a mixture of the four at a fifth site.
Raman spectra recorded at each site enabled rapid identification of each nanoparticle type. Spectra recorded at the fifth site could be analysed to calculate the relative concentration of each type of nanoparticle present. The team also produced Raman images of these areas by raster-scanning the injection sites and recording spectra at 750 µm steps. Algorithms were then used to generate colour-coded, 2D images of nanoparticle concentration.
"Because cells are so complex, almost any application in biology will require multiplexing," Gambhir told medicalphysicsweb. "We have previously been limited to studying one protein at a time, but now we can study multiple proteins. We can't get to thousands of different signals – but 10–30 may be possible."
In another experiment, the team examined the liver pharmacokinetics of pegylated (attached to polyethylene glycol polymer chains) and nonpegylated SERS nanoparticles, each with different Raman signatures, in living mice. The source laser was positioned above the mouse's liver and nanoparticles injected into its tail vein. By acquiring Raman spectra at 30 s intervals, the researchers determined the relative concentration of the two nanoparticle types in the liver, as a function of time.Tumour targeting
The Stanford team has also investigated in vivo imaging of single-walled carbon nanotubes, which have an inherently intense Raman peak. The researchers injected mice with the nanotubes and acquired a raster scan (1 mm step size) over a large area of the mouse body two hours after injection.
The resulting Raman image revealed accumulation of nanotubes in the liver plus a random distribution faintly dispersed across the peritoneal cavity. The signal in the liver increased following injection, with nanoparticles visualized in the liver region up to 12 days after injection.
Furthermore, preliminary data demonstrated the ability to image targeted nanotubes in a mouse-tumour model. Nanotubes conjugated with a tumour-targeting peptide were injected into living mice and raster scans (750 µm step size) acquired 24 h later. Raman images revealed an intense accumulation of the tagged nanotubes in the tumour area. In contrast, little to no accumulation of untagged nanotubes was observed in the tumour region.
This tagging procedure could also be adapted for tumour visualization using the SERS nanoparticles. "The next step is to evaluate the SERS particles' biodistribution in the body by radiolabelling them and using microPET," Zavaleta told medicalphysicsweb. "After we get a good understanding of where these SERS particles naturally end up, we will proceed to tag them (either with peptides or antibodies) for tumour targeting."
Ultimately, Raman spectroscopy could play a diagnostic role within human subjects. The primary limitation here will be the inherent penetration of near-infrared light (no more than a few centimetres in tissue). Gambhir, however, predicts that endoscopy-based Raman spectroscopy may become feasible in about 12 to 18 months' time, for example to detect colorectal cancer in patients undergoing colonoscopy. "We plan to extend both small-animal use and human use with these Raman strategies," he said.