TERS approach curve

QNM data on
PEO-SPP

Application Note #136
Advances in Combined Atomic Force andRaman Microscopy
Atomic force microscopy and Raman spectroscopy areboth techniques used to gather information about thesurface properties of a sample, yet their respective userbase is often quite different. There are many important
application reasons to combine these two technologies,
and this application note looks both at the complementary
information gained from both techniques and how aresearcher having access to a combined system can benefit
from the additional information available.

Raman laser on solid
gold tip

as a function of chemical tip modification (i.e., Chemical
Force Microscopy), which is an approach that can help
characterize the distribution of known chemistries in
specific cases but which has not been shown to provide thegeneral applicability of vibrational spectroscopies.

Atomic Force Microscopy
In atomic force microscopy a sharp probe is brought into
close proximity with a sample and held at that distanceby means of a force-based feedback loop. In addition to
the force on which the primary feedback loop is based,different quantities such as electrical current, surface
potential or specific nanomechanical properties can bemeasured. By scanning tip and sample relative to eachother and measuring these quantities at discrete locations
in a serial fashion, three-dimensional images of selected
sample properties can be created. A generic atomic force
microscope (AFM) setup is depicted in figure 1. The
information that atomic force microscopy can provide
has proven to be extremely useful for scientific researchand industrial applications. They lack however, chemical
specificity. That is, unless tip-sample forces are probed

Figure 1. Shown are the most basic parts of an AFM system, a tip,
raster scan mechanism, and data processing unit.

Raman Spectroscopy
Spectroscopy is the study of the interaction ofelectromagnetic radiation with matter. The most common
kinds are fluorescence, infrared, and Raman.1 The latter two
are vibrational techniques, i.e., the energy of radiation theyuse is sufficient to excite molecular or lattice vibrations.
In a Raman experiment, the sample is illuminated withmonochromatic light and the inelastic scattered lightdetected. If a sample is illuminated with light of a frequency
ν0 most of the scattered light is Raleigh scattered, i.e.
elastically scattered and does not change in frequency. A
small portion, however, is at a different frequency ν1 due
to a change in the polarizability of the illuminated molecule.
This shift is referred to as the Stokes shift if ν1 is redshifted with regards to the incident light, or anti-Stokes if ν1
is blue-shifted.

Gratingslit
HNF

samplelaser

Figure 2. Schematic of a Raman spectrometer. The sample isilluminated by a monochromatic light source. After passing througha filter rejecting the laser light it is dispersed by a grating and imaged
onto a CCD chip.

Raman spectroscopy can provide an inside look
into a variety of material-related phenomena suchas compositional information by analyzing peaks at
characteristic frequencies, can give insight into thesymmetry or orientation of molecules or crystals by using
polarization techniques, or can enable the measurementof stress or strain in a crystal by analyzing the shift ina characteristic Raman peak. As a direct probe of thevibrational structure, it does not depend on the presenceof an electronic state with high fluorescence quantum yield(in contrast to fluorescence), giving it wide applicability as aprobe of chemistry and symmetry.
A Raman spectrometer is often combined with an
optical microscope to take advantage of the high spatial
resolution that a confocal optical setup can offer. The
main components of a dispersive Raman setup are a laser
illuminating the sample, optics to collect the backscatteredradiation, a high efficient laser line rejection filter, and finallya spectrometer consisting of an entrance slit, a diffraction
grating and a CCD camera. A basic schematic of a simple
Raman spectrometer is shown in figure 2.
Optical spectroscopies like Raman and atomic force
microscopy are of a complementary nature. Each techniqueoften does not provide a sufficient enough picture of the
problem by itself but when applied in a co-localized fashion
they become quite powerful, i.e., analyzing the same regionof a sample. By doing so, not only can the question of what
the material in the field of view (FOV) is be answered but
also its physical dimensions and properties can be revealedon the nanoscale. The Raman spectrum shown in figure 3
was taken on one of the layers of a cross-sectioned pieceof packing material for unfrosted animal crackers using

Figure 3. Spectrum (black) of a layer of food packaging material and literature spectrum for Poly-propylene (red).

2

Figure 4. Modulus map of the cross-sectioned food packaging material on left. The outer layers are identified by the Raman data as
polypropylene. The AFM data allow for quantification of the mechanical properties. The cross-sectional plot on the right highlights the drop in
modulus from the outer to the inside layer and reveals some particles in the middle layer exhibiting higher modulus than the matrix.

Bruker’s SENTERRA Raman microscope. By comparing
the spectrum obtained with a literature database, one canclearly identify one of the layers as Polypropylene.

322nm, using the Raleigh criterion: R=0.61∙λ/NA. (Forconfocal fluorescence imaging the point spread function is
reduced yielding: R=0.4∙λ/NA.)

That information in itself is useful but other questions
cannot easily be answered by examining the spectrum,e.g., what are the nanomechanical properties of that layer?
It is often not known outside of the small scanning probe
community that with the advent of Peak Force Tapping™,
AFMs are fully capable of not only providing topographic
information but also of quantifying nanomechanicalproperties such as adhesion and modulus. Using a suitableprocedure, the sample was transferred from the SENTERRA
to the MultiMode® 8 AFM. The resulting modulus datafrom the AFM are shown in figure 4. By combining suitablemodes of the AFM and Raman microscopy we are starting
now to get a clearer picture about the chemical nature andfunction of areas under investigation.

In near-field microscopy, one utilizes the effect that asmall object brought in a propagative field induces anevanescent wave and vice versa. One of the characteristics
of evanescent fields is that they decay exponentially withincreasing distance, thus offering a gateway to resolution
beyond the classical diffraction-based limitations. By using
a suitable AFM tip as a scatter source and subsequently
scanning the sample, an optical map together with
topography and other typical atomic force microscopy
information can be acquired. A general setup for linear
and non-linear tip-assisted spectroscopies is shown infigure 5. Tip-assisted measurements can be carried out intransmission and reflection geometry, thus addressing theneeds to characterize opaque and transparent samples.
Examples for suitable AFMs include Bruker’s BioScope
Catalyst™ and Innova® for transparent and non-transparent
samples, respectively.

The Next Step
The full synergistic effect of optical spectroscopies andatomic force microscopy comes into play when we task
the AFM tip with “becoming” the light source. Lateral
resolution is limited by diffraction and in a conventional
upright Raman setup, is typically 500nm–1µm with a depthof focus of around 1µm. We can calculate as an example,
the lateral resolution in a transmission mode setup with ahigh NA oil-objective of NA=1.2 and red laser light to be

Attempting Raman microscopy on the sub 50nm scalewith a tip that does not enhance the Raman effectsignificantly would be hopeless due to the very low Ramancross section of <1 in roughly every 10 6 photons. If one
chooses, however, a suitable combination of tip andincident light, and places them in the correct geometry, astrong electromagnetic field is generated at the apex of3

Figure 5. General setup for linear and non-linear tip-assisted
spectroscopies.

Figure 6. Field enhancement by the tip.

the tip. This field is mainly due to the incident light excitinga plasmon resonance in the tip. The corresponding dipole
in the tip can even further enhance the field if a suitable
metallic substrate is chosen, as the tip can induce a dipole
in the sample. Suitable metals in the typical tip geometries
are silver using green light and gold using red light as
the excitation sources. The field enhancement by the
tip is twofold as illustrated in figure 6. The tip acts as anantenna to enhance the incident field and as a transmitterto enhance the radiated field. This strong enhancement iswhat makes Raman spectroscopy on the nanometer scalefeasible and creates a whole new area of research. 2

and also limits the lifetime of integrated circuits. The
commonly used far-field Raman measurements canprovide information about stress but the spatial resolution
is restricted to the diffraction limit. Here TERS offers thepotential for a solution by pushing the spatial resolution of
optical spectroscopy into the range of AFM.

It should be noted that there is still some discussion in thescientific community about the enhancement of the field
due to the so called “lightning rod” effect. A thoroughdiscussion would go beyond the scope of this note andthe reader is encouraged to follow the current literature.
Due to the strong localization of the EM field around thetip, TERS exhibits a much higher surface sensitivity than
far-field Raman and is quite similar to SERS in that respect.3
The polarization along the tip axis also has an effect on theselection rules that becomes important when comparing
far-field and near-field data.4
TERS and other tip enhanced spectroscopies open the door
to a whole new field of research. The improvement in spatial
resolution is the obvious gain one expects from combining
traditional far-field spectroscopies with atomic force
microscopy. A difference in selection rules between nearand far-field experiments, as well as a higher sensitivity tosurface features for TERS are just two examples why TERS
will be similar to conventional Raman spectroscopy but will
not yield the exact same information just on a smaller lengthscale. It is expected that due to similarities between SERS
and TERS that the research on TERS will also lead to a
deeper understanding of SERS and vice versa.
In the semiconductor industry, the control of stress inmaterials is a crucial task as it affects device performance4

In the pharmaceutical industry, the occurrence ofpolymorphs, that is the same chemical compositions but ina different crystal lattice, can be crucial for the property of adrug. Raman spectroscopy is used to study polymorphism,
and co-localization with various AFM techniques canincrease the productivity of the research carried out.
Analyzing unknown material is a common task in many
corporate laboratories. This is an important step to ensurethat intellectual property is protected. Co-localization of
AFM techniques with optical spectroscopies is a great stepin speeding up this process.

Solutions for a Combined Approach
The biggest challenge in a combined instrument enabling
Raman spectroscopy and nanoscale surface characterization
is not to compromise the performance of either one. There
are two typical things to consider:
1. To keep the photodetector noise in an AFM low, typicalbeam-bounce systems in an AFM operate in the red witha power of about 1mW, which translates to 3∙1018 photons/
second. This is quite a lot of background considering theineffectiveness of the Raman effect, and at a wavelength
where the often used red HeNe laser of the Raman system
operates. To allow parallel, simultaneous operation of thespectrometer and the AFM, the wavelength of the AFM
beam-bounce system should therefore either be changed to
the near-IR to not interfere with the optical measurements
or a non-optical feedback system such as tuning forks
should be employed.5
2. Spectrometer systems often employ several lasers that
may be cooled by noisy external fans or water coolingFigure 7. View of the Dimension Icon stage and optics arm of the Raman microscope. The Icon stage shuttles the sample between the
AFM head (left) and the Raman objective (right). The red spot emanating the objective is the Raman laser illuminating the sample during a
Raman measurement.

systems or radiate quite some heat in the proximity of
the AFM. Both of these effects can negatively impact
AFM performance. Noise from heating fans can couple
into the AFM and cause instabilities in the feedback loop.
Temperature changes will cause the AFM to drift and will
make it extremely difficult to keep the tip in the selected
field-of-view.
The associated hurdles combining a Raman microscope andAFM actually make another physical solution viable. That isa shuttle stage designed to transfer samples from one tothe other instrument with a possibility to register them to
a common coordinate system. This is not a fancy solution
at first glance but it allows the use of both instruments, theAFM and the Raman spectrometer, to operate without any
compromise in performance. A shuttle stage also has thepotential advantage of allowing for higher productivity as
both instruments can be used simultaneously.
Where for purely co-localized measurements a tip- or
sample-scanning AFM could be employed, the restrictions
for TERS are more stringent. The laser beam exciting theplasmon resonance in the AFM tip has to stay located on
the tip during the imaging process. That pretty much takes
any tip-scanning AFM out of the equation, as the tip would
leave the laser focus, thus requiring a sample-scanning AFM
such as Bruker’s Innova or Catalyst.
Productivity is one of the main drivers of a co-localized
setup, thus highlighting such features as ease-of use,
availability of various AFM modes, quick sample access andexchange, and ease of calibration of the Raman microscope.
A co-localized system is an instrument that is going to be
used by microscopists and spectroscopists and therefore
should allow for straightforward acquisition of AFM
and optical data as well as a simple and intuitive way ofcorrelating AFM images and optical data.

Figure 8. The Bruker BioScope Catalyst AFM shown with a Renishaw
inVia Raman microscope.

A solution combining Bruker’s industry-leading Dimension
Icon® with Horiba’s LabRam achieves the co-localization ofdata and ensures the high performance of both systems.
The two systems were mechanically coupled by utilizing the
precision of the Icon stage to shuttle the sample between
the AFM and Raman microscope. Figure 7 shows the
combined instrument with the sample in the position for (a)AFM imaging and (b) Raman imaging.
Bruker has also made great strides in enabling instantresearch-quality AFM data for new researchers byintroducing ScanAsyst ®, which almost completely
5

Figure 9. AFM-Raman acquisition sequence with (left) bright-field optical image, (middle) Raman map, and (right) AFM sample
topography image.

automates AFM operation without scarifying performance.6
The following section discusses some results fromco-localized measurements.
Another solution for operation with transparent samples
is the integration of Bruker’s BioScope Catalyst AFM
with a Raman spectrometer shown in figure 8, whichallows for straightforward sample handling without
the need to transfer the sample between methods forco-located measurements.
The Catalyst is an AFM that is fully integrated with aninverted optical microscope. This not only allows thesystem to work in AFM and Raman modes but also to utilize
standard optical techniques, such as phase contrast tointerrogate the sample. The combination, together with the
IRIS software facilitated switch between AFM and Raman
modes, enables high productivity in an open, easy to accessresearch platform.

Co-Localized AFM and Raman Measurements
Example One

compared to the non-aromatic reference line at 1014 cm -1
are lower in the thicker parts of the sample. The Raman
map in the middle of figure 9 shows this strikingly. Raman
data can provide information about the steric orientation ofa sample. Here, the molecules are likely oriented differently
in the thick and thinner areas of the sample. The incident
polarized laser used might therefore help to highlight theorientation of the aromatic rings on the aluminum substrate.

Example Two
Polymorphism is the ability of a material to exist in morethan one crystal structure. This can have profound impacts
on its mechanical behavior or physiological impact in
the case of drugs, for example. The following example
describes a study on Yttria-stabilized zirconia polycrystals(Y-TZP). Y-TZP is often used in dental implants for its
biomechanical and esthetic properties. The material istypically sintered from a fine powder and can be crystallized
in the tetragonal form. An unwanted phase transformation,
however, can take place under certain conditions resulting ina monoclinic lattice.

The following section discusses some results fromco-localized measurements. The first shows an epoxy
compound on a metal substrate. The analysis would
start with the selection of an area using e.g., regularbrightfield contrast. AFM and Raman data can be acquiredin the order selected by the user. Figure 9 depicts such asequence. The bright-field optical image is shown on theleft and the sample topography as acquired by the AFM isshown on the right. The Raman map in the middle of thesequence is based on integration of a user selected area ofthe spectrum.
The two spectra in figure 10 were taken at locations ofdifferent heights of the sample. One can see that thehighest intensity phenyl-ring vibration at 1004 cm -1 can befound in the lower areas of the sample whereas intensities
6

Figure 10. Two Raman spectra taken from sample in figure 9.

The two spectra depicted in figure 11 were acquired on
different locations of a sample. The peaks on the spectra
shown in red can be attributed to Y-TZP. The blue spectrum
clearly shows additional peaks. After subtraction to isolate
the additional peaks and comparing them to literature data,
they can be assigned to the monoclinic phase of ZrO2.
Using DIC optical contrast, an area of the sample was
identified that exhibits two patches of distinctively
different surface morphology. While these patches canFigure 11. Raman spectra of ceramic sample. Red curve shows only
Y-TZP while green curve suggests presence of additional phase.

be described as smooth and rough by looking at the
optical image, the AFM can go further and quantify theroughness. The average roughness of the smooth region
turns out to be 8.7nm, whereas the rough region averages
15.7nm. Automated analysis functions of the AFM allow
for further analysis. Grain size may play an important rolein the transformation from tetragonal to monolclinic. Onceacquired, grain sizes can be extracted from the AFM data.
Figure 12 shows an 83x83um2 DIC image of a smooth andrough patch and a 10x10um2 AFM image of the smooth areaof the sample highlighting the grains. Analysis yields anaverage grain size of 0.56um2.
With the mapping capability of the Raman microscope, one
can now produce a map of an area exhibiting the previously
topographically characterized smooth and rough areas. Oncea map of Raman spectra is acquired, the Raman software
allows one to plot the integrated intensities of a userselected area. In this example, an area from 180–184cm -1
was selected, as this area highlights a peak only present
for the monoclinic phase. By representing the intensities
in a suitable color scheme, a two-dimensional Raman mapof the monoclinic and tetragonal occurrence can thus begenerated. The map and corresponding DIC image areshown in figure 13.

Figure 12. DIC and grainsize data. The optical image (left) depicts a smooth and rough patch on the sample. AFM data (right) of the smoothregion provides grain size detail after automated grain recognition and analysis.
7Figure 13. DIC and Raman map.

The example of performing a combined AFM and RAMAN
analysis of the ceramic sample can be summarized asfollows. The monoclinic to tetragonal phase transformationof Y-TZP results in a surface roughening that occurs

nonuniformly over the surface. The use of co-localized
AFM, Raman, and DIC microscopy enabled the study of theprocess on the nanometer scale.

Figure 14. TERS-ready combination of the Bruker Innova Scanning Probe Microsope and the Renishaw inVia Raman microscope. The optical
coupling is achieved via a trackball operated sampling arm.
8

A Setup for TERS: Innova IRISTERS requires the tip to be as close to the sample aspossible without affecting the integrity of tip or sample. In
addition, a metallic tip is necessary for the enhancement.
STM provides a convenient way to combine theserequirements and study the effects of various tip shapes,
coupling mechanisms, and other variables. The analysis
of opaque samples creates a challenge as high-NA oil
immersion objectives cannot be used, as in transmission
mode setups. A parabolic mirror approach would provide ahigh NA of 1 but puts a lot of constraints on the setup andflexibility. A good compromise and excellent way to excite
the AFM tip and collect the Raman signal is to place theRaman objective at about a 60 degree angle with respect
to the tip axis. It seems counter intuitive but the sideillumination scheme has shown the highest enhancement
factor for TERS in theoretical studies.7-8 A setup using thisside-on geometry is realized in the Innova IRIS shown infigure 14. The Innova lends itself as a platform for TERS on
opaque samples due to its openness, its very stable andlow-noise closed-loop feedback system, and its near-IR
feedback diode. It can be operated in STM and a variety of
AFM modes with easy switching.
Here the Innova is combined with Renishaw’s inVia
microscope to enable TERS, confocal Raman, andco-localized measurements.

IRIS enables TERS measurements in AFM modes and in
STM. The control of both the AFM and Raman microscopeis handled by a software package residing on the AFM
computer. An example of a TERS dataset achieved withsuch a setup is given in figure 15. The sample used is
Malachite Green, a dye for which literature data exist.
Single-molecule sensitivity on Malachite Green using a sideillumination scheme was previously reported by Neascu etal in 2006.9 Spectra like the one presented can be acquiredin as little as 0.1s using just a few micro-watts of incident
laser power.

Conclusion
The existence of co-localized instrumentation now
allows researchers to interrogate samples using optical
spectroscopy and scanning probes techniques yielding
detailed information about nanoscale properties andcomposition. Bruker provides solutions for opaque andtransparent samples with the Dimension Icon, BioScope
Catalyst, and Innova systems. TERS promises to push theresolution limits even further and enable the collectionof chemical information on the nanometer scale.
Bruker’s solutions for this advanced research include
the Catalyst and Innova for transparent and opaquesamples, respectively.

Figure 15. TERS spectra of Malachite Green obtained using a gold tip
illuminated by 633nm light at varying distances above the surface.
Data acquired using the IRIS Innova-InVia combination. By comparing
the peak intensities with the tip approached to the retracted spectra,
one can clearly see the enhancement of Raman modes.

9References
1. H. Kuzmany, Solid State Spectroscopy, 2nd edition (Springer 2009).
2. R. Stöckle, Y.D. Suh, V. Deckert, and R. Zenobi, Chem. Phys. Lett. 318,
131 (2000).
3. T. Ichimura, S. Fujii, and S. Kawata. Phys.Rev. Lett. 102 (2009).
4. C.C. Neacsu, S. Berweger, and M.B. Raschke, Nanobiotechnology 3, 172(2007).
5. W.H.J. Rensen, Ph.D. thesis, Univ. of Twente (2002).
6. S.B. Kaemmer, Bruker AN133 (2011).
7. A. Downes,D. Salter, and A. Elfick, J. Phys. Chem. B 2006, 110,8. Z. Yang, J. Aizpurua, and H. Xu, J. Raman Spectrosc. 2009, 40,©2012 Bruker Corporation. All rights reserved. BioScope Catalyst, Dimension Icon, Innova, IRIS, NanoScope, and TappingMode are trademarks or

AuthorStefan Kaemmer, Ph.D., Bruker Nano Surfaces Division(stefan.kaemmer@bruker-nano.com)

Acknowledgements
YZTP sample courtesy of Dr. Ing. Lubica Hallman (Center of Dental Medicine,
University of Zürich).
I would like to acknowledge the help from my colleagues: Ton Ruiter, Ph.D.,
Natalia Erina, Ph.D., Igor Nemeth, Ph.D., and Tim Batten, Ph.D. (Renishaw).

Bruker Nano Surfaces Divison
Santa Barbara, CA · USA+1.805.967.1400/800.873.9750
productinfo@bruker-nano.com

www.bruker .com

registered trademarks of Bruker Corporation. All other trademarks are the property of their respective companies. AN136, Rev. A2

9. C.C. Neascu, J. Dreyer, N. Behr, and M.B. Raschke, Physical Review B 73,
193403 (2006).