www.rdmag.com August 2014 R&DMagazine 9
and reproducible tips in the laboratory.
Horiba Scientific, in turn, is working closely with SPM specialist AIST-NT, Novato, Calif.,
to increase the capabilities of its TERS-ready
microscopes, which include the compact, automated XplorRA nano, broad wavelength HR
Evo Nano and bioresearch-ready CombiScope
XploRA. Each are equipped with AIS T-NT’s
SmartSPM for AFM-Raman studies. Horiba
has added a side port to its microscopes, along
with high 0.7 NA objectives, to allow customers to use polarized lasers to achieve maximum
AFM modes contribute their part
Many major microscope vendors have
launched systems intended for combined
AFM-Raman nanoscale research. They include
Thermo Fisher Scientific, JPK Instruments,
Asylum Research and Nanonics.
One vendor, WITec GmbH, Ulm, Germany,
has based much of its product line on confocal Raman microscopy, and has added AFM
to many of its instruments. The approach,
which helped earn the company a 2008 R&D
100 Award, reflects an increasingly modular
approach to instrument construction and
“The advantage of the WITec systems is that
all imaging techniques are truly integrated in
a single instrument with one controller and
software,” says Dr. Sonja Breuninger, technical
marketing director for WITec. “The setup is
highly flexible and can be upgraded or complemented when required. Imaging techniques
such as NSOM or scanning electron microscopy (SEM) can be easily added.”
A WITec microscope can be as basic as
the alpha 300 M for single Raman spectrum
measurements or as complex as the alpha 300
RAS, equipped with combined Raman, AFM,
NSOM, fluorescence and luminescence imaging techniques. This adjustability, says Breuninger, pays dividends in biological research
where analysis conditions change frequently.
Software is a particularly important addition
in this field, she adds, because it can generate
depth profiles and 3-D images, even of soft
At Bruker Nano Surfaces, developers have
streamlined AFM functionality directly into
an existing inverted research microscope.
According to Andrea Slade, AFM applica-
tions specialist at Bruker Nano Surfaces, the
introduction of Bruker’s Integrated Raman
Spectroscopy System (IRIS) TERS probes in
2010 have generated more than 330 scientific
papers. Of these, 110 were directed at biologi-
cal or bio-related research, followed closely by
detailed study of polymers.
“In chemistry and biology, many techniques
complement each other. Raman spectroscopy
complements direct vibrational spectroscopy
very well,” says Slade, which is why Bruker
launched the IRIS technology. “There’s an
increasing interest in AFM activities, or modes,
in conjunction with Raman and fluorescence.
A lot of this work is physics oriented, but
biologists have experimented and developed
bio-applications that work.”
It’s still a challenging technique, she adds,
and requires more finesse. “It involves having
the right tips and probes, and having to deal
with sample restrictions,” she continues.
To facilitate the addition of scanning probe
analysis, IRIS-ready instruments have been
designed with open optical heads to avoid
interference. AFM mode development is
proceeding quickly at Bruker, which has pioneered several new techniques in recent years.
Arguably the most influential new mode is
Peak Force Mapping (PFM), which allows
researchers to perform studies on biological
tissues such as amyloid fibrils, which can have
deleterious effects on brain cells. Using PFM,
researchers can accurately determine a cell’s
modulus and what type of force allows the
fibril to pass through a cell membrane. Hoogenboom and Leung relied on PFM for their
recent DNA research.
A new variant of Bruker’s PFM, called
Peak Force Mapping QNM (Quantitative
Nanomechanical Property Mapping), permits
quantitative mechanical mapping of material
properties while also imaging sample topography at high resolution. QNM works by
oscillating the probe at resonant frequencies
of 1 or 2 kHz to allow imaging of soft samples.
In tapping mode, researchers can use the AFM
tip to “push” on samples. The system addresses
an existing problem with using resonance to
image fluids. In tapping mode, the user must
continuously tune the probe to achieve the
resonant peak. This can’t easily be done for
liquid samples. QNM instead feeds back on the
applied force between the tip and the surface,
and that force is always precisely known. When
used with Bruker’s Scan-Asyst technology, the
set point self-optimizes, allowing more consistent results on the sample.
“QNM is being used now to look at glio-blastomas, and whether they are stiffer than
healthy counterparts, or are less stiff and more
deformable. This information is important
because it gives researchers an idea of how cancer cells metastasize,” says Slade.
In the case of Hoogenboom’s DNA analysis
work at the London Centre of Nanotechnology, the AFM probe tip on the FastScan Bio
can determine minute changes in the topography of DNA molecules. By looking at the
force-distance curves generated by the AFM
and comparing them to the Raman spectra
collected by the microscope, the researchers
can build a database of relational data that can
help inform scientific studies.
Combined systems to aid
Only 12 years old, TERS as a laboratory imaging technique is still in its infancy. Some scientists have eagerly explored the new limits the
technique offers, but it remains out of reach
for most. Major global research efforts like the
BRAIN Initiative will continue to rely on targeted fluorescence-based techniques for discovery. But Slade says that Bruker’s customers
now generally recognize the advantage of AFM
as a complementary microscopy technique,
and that an understanding of “
mechano-biology” will be important going forward.
The possibilities are substantial: In 2013, a
team led by Zhenchao Dong at the Univ. of
Science and Technology in China combined
AFM-Raman with a scanning tunneling
microscope to successfully map an individual
molecule at a resolution of less than 3 nm.
Bruker’s efforts to improve performance have
resulted in the first TERS probes designed specifically for one system, the Innova-IRIS AFM.
Image: Bruker Nano Surfaces