8 R&DMagazine August 2014
A New Spectrum
Raman spectroscopy has long been a powerful tool for biological research. The addition of
atomic force microscopy is adding an important new dimension.
In 2012, a team of researchers in London imaged, for the first time, the structure of the DNA double helix. James Watson and Fran- cis Crick discovered DNA 60 years ago by
laboriously studying x-ray diffraction images of
millions of DNA molecules. However, Dr. Bart
Hoogenboom and Dr. Carl Leung used atomic
force microscopy (AFM) to directly “feel” the
molecule’s structure in a fraction of the time.
Since then, the team has taken AFM further,
conducting a single-molecule analysis of the
secondary structure of a biomolecule. This
ordinarily requires crystallization, but was, in
this case, done with a conventional sample at
room temperature. Their tools included Multimode 8 and FastScan Bio AFM systems from
Bruker Nano Surfaces, Santa Barbara, Calif.
They also used a Cypher microscope from Asy-
lum Research, Santa Barbara, Calif., equipped
with biolever mini-cantilevers from Olympus
America, Center Valley, Penn., to conduct
amplitude-modulation experiments. With these
tools in hand, they accurately reproduced the
dimensions of an important—and extremely
small—DNA crystal structure.
This leap in accuracy is the result of a marriage between AFM and Raman spectroscopy.
These techniques have been used together in
materials science for a number of years to map
surfaces and acquire chemical information.
More recently, scientists working in bio-related
fields are recognizing its potential. And instrument developers are responding by introducing new capabilities for imaging biological
systems in unprecedented detail.
Raman enters the nanoscale realm
For many years after its discovery, Raman
spectroscopy was held back by the diffraction
limit of light, which limits vibrational spectroscopic acquisition, and the chemical information this data contains, to 250 nm. Instead
biologists looked to other methods, such as
electron microscopy and near-field optical
microscopy (NSOM), a form of scanning
probe microscopy (SPM) that combines optics
with a probe that reads evanescent waves. In
recent years, fluorescence-based techniques
have allowed vendors to market super-res-olution optical microscopes that peer deep
into the nanoscale. And in the Raman space,
a technique called surface-enhanced Raman
scattering (SERS) takes advantage of small-scale electromagnetic effects to “boost” the
Raman scale by a factor of 10 or more, helping
Raman surpass the diffraction limit.
But SERS isn’t capable of localization. To
understand precisely the position and activity
of individual nanoscale biological features,
namely cells and proteins, researchers need
the ability to map, or scan, extremely small
regions. Tip-enhanced Raman spectroscopy
(TERS) provides this capability by shrinking
the sample to the size of a cantilever tip.
“With Raman, you have imaging capability
down to 250 nm, which is about 200 times
less resolution than with AFM. At this point
TERS comes into play. The typical approach is
to use aperture probes to beam lasers through
fiber or a hollow cantilever to get a small spot
size on a sample,” says Emmanuel LeRoy,
AFM-Raman product manager at Edison,
N.J.-based Horiba Scientific. “ This doesn’t
work well with Raman because the scattering is
weak. Only one in a million photons are scat-
tered back and, because the volume is so small,
no Raman signal is returned.”
The probe itself supplies the enhancement,
which is localized at the bottom of the probe,
in an area smaller than the tip. With TERS
probe tips, the tip size is 20 to 30 nm, provid-
ing a localization of just 5 to 10 nm.
This sort of capability has attracted the
attention of biophysicist Volker Deckert, a
professor at Jena Univ. in Germany who has
successfully used Horiba Scientific’s TERS-ca-pable instruments to conduct studies of biological constructs such as DNA origami.
“TERS allows structural investigations with
an impressive lateral resolution,” says Deckert.
“The benefit is two-fold. First, the driving force
behind the development of this technique was,
and still is, our general interest in molecular
structures below the diffraction. Second, AFM-based vibrational spectroscopy techniques, and
TERS in particular, are best suited when the
use of additional labels is prohibitive or simply
In addition, adds Deckert, it’s always desirable to perform work on “real” systems. Acquisition of chemical data from living systems can
be done with high lateral resolution, in real
time and label-free. TERS has allowed him to
study complex samples such as amyloid fibrils,
cellular proteins and even crystal structures
produced by the malaria parasite. “A vast
number of scientific questions can now be
addressed,” says Deckert.
TERS isn’t perfect for biology, however.
Like any SPM-based technique, it’s limited to
surface analysis. Also, LeRoy says, the method is comparatively slow: Only a handful of
molecules are studied at any one time, which
reduces Raman signal. Plus, biological samples have a tendency to “stick” to the probe
tip, which can complicate some soft tissue
analysis. Finally, the lack of highly developed
probes held back research because a broken or
damaged tip couldn’t easily be replaced. This
is being rapidly corrected, however, as scientists like Deckert are developing more efficient
When employed in instruments like Horiba’s
XploRA Nano, TERS can perform comprehensive measurements of nanoscale surfaces,
including biological specimens.