Gary Boas, News Editor, email@example.com
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
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
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
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
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
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]
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
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.