2Introduction to scanning near-fieldmicroscopy
CHAPTER 2
2 Introduction to scanning near-field microscopy
2
2.1 Introduction
This chapter is meant to provide a short summary of the techniques that have been
used in this work, which gravitates around the idea of combining scanning probe
microscopy (SPM) with optical techniques: atomic force microscopy (AFM) (sec-
tion 2.2), vibrational spectroscopy (section 2.3), and scanning near-field optical mi-
croscopy (SNOM) (section 2.4).
2.2 Atomic force microscopy (AFM)
The history of AFM dates back to 1982, when IBM researchers introduced scanning
tunneling microscopy (STM) [1]. This technique uses a small tunneling current
between a nanometer sized metallic tip and a conducting sample to control the
separation between tip and sample. The tunneling current exponentially decays with
gap distance over less than 10 nm, and is therefore a very sensitive control mechanism.
As the tip is scanned over the sample, a piezoelectric stage adjusts the relative
position of the two to maintain the current constant. Therefore, the voltage applied
to the piezoelectric stage directly provides an image of the surface of the sample with
a spatial resolution down to the atomic level [2–5]. STM only works with conducting
samples and clearly fails in liquids. To overcome this issue, the same researchers
proposed to replace the metallic tip with a cantilever spring, equipped with a sharp
pyramid underneath its free hanging end, as illustrated in Fig. 2.1a [6]. As the tip
hovers over the surface, the attractive and repulsive forces with the sample make
the cantilever bend and enable measurements on almost any surface. The cantilever
deflection is monitored by a laser beam reflecting on the cantilever and recorded
by a position sensitive photodetector. This configuration amplifies the cantilever
movements and enhances the sensitivity to the sub-nanometer level. Maintaining
the signal registered by the photodector constant, one can thus keep the distance
between the tip and the sample constant without a tunneling current – an approach
that has been dubbed as atomic force microscopy. Over the last 15 years, AFM
has developed from a specialized research tool requiring a highly trained operator to
a frequently used machine in a wide variety of research fields [7–9], including cell
biology, molecular biology, material sciences and graphene research.
2.2.1 Working principle
There are three main operation modes: (i) contact mode, (ii) non-contact mode,
and (iii) tapping or intermittent contact mode. The three force domains where these
12
2.2 Atomic force microscopy (AFM)
2
a)
Tapping-mode
Distance
Forc
e
Repulsive
Atractive
Contact
Non-
cont
act
b)
Figure 2.1: Illustration of atomic force microscopy and the optical triangulation method(a) and the three AFM operation modes and their force domain (b).
modes operate are shown in Fig. 2.1b. In contact mode, the tip is kept in physical
contact with the surface throughout the scan and dragged over the surface; keeping
the signal measured at the photodetector constant, one can thus acquire an image
by recording the voltage applied to the piezoelectric element that moves the base of
the cantilever (or the sample stage) vertically. In non-contact mode, the cantilever
is made to oscillate at very close separation with respect to the sample (15 - 50 A)
without ever making contact with the surface; the long distance attractive van der
Waals forces induce changes in the resonance frequency and oscillation amplitude
– a piece of information that can be used in the feedback mechanism to keep the
tip-sample distance constant during scanning. Tapping mode is similar to amplitude
non-contact mode, except that the cantilever does enter into contact at the end of
every oscillation period. Tapping mode is nowadays the most versatile and, therefore,
most used imaging method [10]. This technique uses the most robust distance control
mechanism and therefore also adapts well to soft or sticking samples. However, for
very fragile samples, applications in liquids, or samples that are characterized by
strong adhesion forces, one may want to resort to one of the other approaches. For
instance, contact mode is particularly adapt to measurements in liquids, although it
can damage the sample; non-contact mode is an excellent imaging method on well
defined surfaces in air or vacuum, but may be more difficult to use with extremely
rough samples, in the presence of high capillary or electrostatic forces, and in critical
environments.
2.2.2 Recent applications and limitations relevant to this
work
AFM was developed to make high-resolution imaging available for any kind of sample.
Resolution and speed therefore, are the most important properties that have been
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2 Introduction to scanning near-field microscopy
2
improved significantly over the last decades. Pushing the limits of the technique, re-
searchers have recently demonstrated that AFM can directly visualize structural and
functional details that, before, could only be shown with indirect methods, includ-
ing nuclear magnetic resonance (NMR), X-ray crystallography, or vibrational spec-
troscopy. Ultra high-resolution AFM now allows for true atomic resolution imaging.
Some studies have shown that AFM can make the structure of organic molecules
visible [11, 12]. Other researchers have reported that it is possible to study reaction
mechanisms of organic molecules [13, 14] or to investigate self assembly of 2D DNA
crystals [15] using AFM imaging. These kind of experiments can only be done on
extremely clean and flat surfaces and at very low concentrations of analyte to ensure
that molecules do not overlap, but they are possible.
As with most scanning techniques, a major drawback of atomic force microscopy is
the imaging speed, in particular in applications that aim to follow dynamic processes.
A typical scanspeed for a 10 X 10 µm2 image is around 1 Hz, which means that
scanning this area with a resolution of 1024 X 1024 pixels would require 17 minutes.
Faster systems require faster electronics, faster piezoelectric elements, and special
cantilevers with high resonance frequencies of more than a MHz. An increased speed
therefore goes at the cost of resolution or scan range. The feedback mechanism is
often the limiting factor. A simple calculation as demonstrated by Toshio Ando [8]
can show the required time constant to be able to reach a certain imaging speed.
The fastest general use, commercially available AFM is the Bruker Dimension Fast
Scan that reaches acquisition rates up to 4.6 frames per second for very small sample
areas (area: 100 nm2, 128 x 64 pixels). Typical timescales for biological processes
are in the sub-100-millisecond to one second range. Protein folding, DNA or cell
morphology dynamics are a few examples [8, 16, 17]. The AFM movie of walking
Myosin V motor protein is also very famous for this reason [18] as it shows life in the
greatest detail currently available.
The AFM is an excellent instrument for imaging nanoscale features of small
samples. However, this data is limited to information about the surface and provides
limited information about the chemical nature of the substance. A useful addition
would be the possibility to simultaneously gather optical information on the point
scanned by the tip – a problem that is partially solved by SNOM and, in part, by the
work of this thesis.
2.3 Vibrational spectroscopy
The interactions of light and matter, or spectroscopy, provide valuable information
about the chemical nature of a sample. We can differentiate between different
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2.3 Vibrational spectroscopy
2
types of spectroscopy, such as absorption, fluorescence or Raman spectroscopy (see
Fig. 2.2). In this section I will only focus on vibrational spectroscopy because it
does not require labels and provides the most detailed molecular information. In-
frared (IR) and Raman spectroscopy are the two most commonly applied groups of
vibrational spectroscopy. In IR, infrared light with an energy matching a vibrational
transition of a molecule is absorbed by a sample. The specific absorption spectrum
can, for example, be used to identify a substance. This technique, however, has the
disadvantage of requiring long infrared wavelengths that limit the lateral resolution
in microscopy. Raman spectroscopy, on the other hand, provides the same informa-
tion using a visible excitation source with sub-micrometer wavelengths and therefore
allows for diffraction limited imaging at a resolution of 200 - 300 nm.
2.3.1 Raman spectroscopy
The Raman effect was first observed by Indian scientists C.V. Raman and K.S. Kr-
ishnan [19]. They discovered in 1928 that filtered sunlight focused on a sample also
returned some light with a shifted frequency compared to the incident beam. Sixty
different liquids were tested and all returned a very specific frequency range or spec-
trum, which can be understood as evidence for interactions between light and the
sample. At that time the laser was not yet invented and spectrometers did not exist,
thus only color filters could be used to measure the frequency shift.
After the invention of the laser, Raman spectroscopy quickly became more popular
and nowadays it is a widely used and versatile research tool. The specific molecular
information, hidden in Raman spectra, makes it an excellent technique for studying
dynamic processes [20–22].
2.3.1.1 Principle
In Raman spectroscopy a sample is illuminated by a monochromatic laser beam that
interacts with the molecules in the sample. Excitation to a so called virtual state
(dashed line in Fig. 2.2) leads to inelastic scattering of photons, which means that the
optical beam polarizes the electron cloud while energy transfer between the light and
the molecule takes place. The extra energy is used to excite one of the molecular
vibrations, which thus results in the scattered photon having a lower energy than
the incident photon. When energy is transferred to the molecule the scattering
event is called Stokes Raman. The opposite process is however also possible. In
anti-Stokes Raman scattering, already excited molecules lose energy to the optical
beam. The energy differences between the incident beam and scattered photons
forms a Raman spectrum that is specific for the present substance or mixture. The
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2 Introduction to scanning near-field microscopy
2
Ram
an
hν
S0
S1
Stokes Anti Stokes Rayleigh InfraredResonance Raman
Figure 2.2: Jablonski diagram showing Raman and Rayleigh scattering and infrared ab-sorption spectroscopy.
Boltzmann distribution implies that the majority of molecules will be in the ground
state at standard conditions, therefore, it is clear that the Stokes spectrum is much
stronger than the anti-Stokes spectrum.
2.3.1.2 Surface enhanced Raman spectroscopy (SERS)
A major drawback of Raman spectroscopy is the extremely low scattering cross sec-
tion of about 10-31 - 10-29 cm2 per molecule, which, for example, is about 14 orders of
magnitude lower than the fluorescent cross section of lightly fluorescent compounds
[23]. The scattering probability is related to λ−4 and thus the signal can be improved
using a shorter excitation wavelength although this has the risk of a higher fluorescent
background. Shorter excitation wavelengths can however not always be used, since
photo bleaching, due to high photon energies, is more likely to occur. In resonance
Raman spectroscopy (RRS), the excitation wavelength is tuned to a molecular ab-
sorption line, which results in a selective signal enhancement for chromophores that
can even be as high as 1014 for special conditions. This method is especially useful
for larger biomolecules, such as proteins, to obtain information, only about certain
functional parts of the molecule [24, 25].
Another way of signal enhancement is surface enhanced Raman spectroscopy
(SERS), which was first reported by an early paper of Fleischmann et al. in 1974
16
2.3 Vibrational spectroscopy
2
[26]. In this work, pyridine molecules were adsorbed to a roughened silver electrode,
which induced strong enhancement of certain Raman bands. Since that time, SERS
became a very important technique in many fields of research, including biology [27],
catalysis [28] and environmental sciences [29]. Typical substrates used in SERS are
roughened metal electrodes, nanostructured surfaces or metal nanoparticles. For dye
molecules very large enhancement factors of 1010 to 1011 have been observed [30,
31], which means that the effect is much closer to that of fluorescence.
The exact mechanism explaining the large enhancement factors is still not fully
understood. However, two main contributions are generally accepted, the electro
magnetic mechanism and the chemical mechanism.
Electromagnetic theory
The electromagnetic enhancement is generated by the excitation of localized surface
plasmons of a metal substrate. Surface plasmons are coherent delocalized electron
oscillations that exist at the interface of a conductor and dielectric (for example a
metal and air). These surface plasmons can be excited when the wavelength matches
the energy of the plasmon oscillations. When a metal surface is nano-structured, for
instance in the form of metal nanoparticles, the surface plasmons are trapped around
the particle as localized surface plasmons (LSP). The LSP resonance frequency de-
pends on the material and particle size. Excited LSP, known as surface plasmon
polaritons, create a very strong local electromagnetic field that decays exponentially
with distance. Even stronger fields can be created when the LSP’s of two particles
overlap and create a so-called gap mode. The field enhancement can be modeled
using Mie scattering theory, as shown in Fig. 2.3 for a single and double gold sphere
[32].
a) b)
1
10
1
100
Figure 2.3: Mie scattering simulation of a 30 nm gold sphere (a) and two 30 nm spheresclose together (b). Taken from Ref. [33].
The electromagnetic field enhancement I is related to the Raman enhancement
by the electric field E and the wavelength λ through the relation:
I ∝ E2
λ4(2.1)
17
2 Introduction to scanning near-field microscopy
2
Hence, the Raman intensity is inversely related to the fourth power of the wavelength
and proportional to the quadratic power of the electromagnetic field. Therefore, the
highly intensified electromagnetic field close to the scattering particle will cause larger
Raman enhancement [33]. This SERS effect decays by a factor RR+d
for a molecule
with distance d away from a particle with radius R.
Overlapping fields between two materials can excite the gap mode that makes
the resonance frequency shift.
Chemical theory
Although the electromagnetic theory can be applied independent of the chemical
system of interest, it does not fully explain the large enhancement factors observed
for particular classes of compounds. The chemical theory only applies to certain
chemical systems and involves charge transfer between the molecule and the metal
surface. The mechanism is comparable to resonance Raman enhancement. A strong
electromagnetic field, as in the molecular excited state, can induce charge transfer
from the metal particle to the analyte, which forces an electron to move to the
lowest unoccupied molecular orbital (LUMO) [34]. To profit from this mechanism,
chemical interactions between the molecule and metal surface are required. This can
be achieved through functional groups such as thiols and amines that have a high
affinity for metal substrates.
2.3.1.3 Tip enhanced Raman scattering (TERS)
Tip enhanced Raman spectroscopy (TERS) works according to the same principle as
SERS with the subtle difference that TERS is a single hotspot technique and poten-
tially provides high-resolution spatial information when the position of the hotspot
can be controlled. The apex of a metalized AFM or STM tip can function as a
plasmonic nanoparticle and causes a strong signal enhancement (see Fig. 2.4). The
enhancement factor is large enough to reach sufficient sensitivity to enable very high-
resolution, and sufficiently fast nanoscale imaging. The combination of spectral and
spatial information makes it an important method for many nanoscale applications.
Over the last 15 years this technique has gained a lot in popularity, and has grown
in functionality and applicability. However, TERS systems often remain complicated
and stable high signal enhancement factors are not easy to obtain during imaging.
Further details are beyond the scope of this chapter. Three recent papers about the
developments in TERS can be found in [35–37].
18
2.4 Scanning near-field optical microscopy (SNOM)
2
Figure 2.4: Illustration of tip enhanced Raman scattering.
2.4 Scanning near-field optical microscopy
(SNOM)
Scanning probe microscopy (SPM) is a general term for tools that can provide more
than structural information, extending AFM with other sample properties, includ-
ing stiffness and electrical characteristics. Scanning near-field optical microscopy
(SNOM) is a SPM method with which optical information at the nanoscale can be
obtained.
SNOM overcomes the fundamental diffraction limit using the near-field for imag-
ing [38–41]. The near-field can best be described as the non-propagating electromag-
netic field exiting from an emitting object, which decays exponentially with distance
and reaches into space for a distance in the order of a wavelength. It can thus only
be detected by a detector at close proximity to the emitter. Hence, the maximum
achievable resolution is defined by the size of the detector or source rather than the
wavelength of the used radiation for example.
In SNOM, a nanoscale tip functions as a small detector or light source and is
scanned across a sample while recording optical information. This technique can be
divided into two main groups of instruments: aperture SNOM [42, 43] of which the
diferent operation modes are shown in Fig. 2.5 and apertureless SNOM [44–46].
19
2 Introduction to scanning near-field microscopy
2
a) d)c)b)
Figure 2.5: Different aperture SNOM operation modes. Excitation (a), collection (b),reflection (c), and cantilever probes (d) can also be used in all modes.
2.4.1 Apertureless SNOM
Apertureless or scattering-type SNOM uses a metal tip that functions as an antenna
to specifically excite the molecules close to the tip apex. LSP resonances induce large
signal enhancements for molecules under the tip. This mechanism can, for example,
be used in near-field infrared imaging as in scanning near-field infrared microscopy
(SNIM) or in combination with Raman spectroscopy as with TERS, which is briefly
described in section 2.3.1.3.
2.4.2 Aperture SNOM
In Aperture SNOM an optical fiber is drawn or etched into a sharp tip, which functions
as a tiny detector or light source. In this technique the lateral optical resolution is
defined by the size of the aperture at the tip apex rather than the wavelength.
Currently, a spatial resolution of about 50 nm can be typically obtained.
In 1928, Synge [47] proposed a method to acquire images with a resolution beyond
the diffraction limit. The concept is straightforward. If one brings a light source with
dimensions much smaller than the wavelength close to a sample, a sub-wavelength
spot will illuminate the surface.
The principle of aperture SNOM was well explained by Pohl et al. [48]. In that
work, the metaphor of a doctor’s stethoscope was used to explain the technique. The
location of the heart can be found with a precision of less than 10 cm by moving
the stethoscope over the chest. The calculated resolution, using this technique, is
almost λ/1000, where λ represents the wavelength of the sound wave (speed of sound
through bone is about 3000 m/s). The principle of an aperture SNOM measurement
is, in fact, very similar. A very tiny light source or detector is moved over a sample to
acquire an image with a resolution equal to the size of the scanning object. To avoid
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2.4 Scanning near-field optical microscopy (SNOM)
2
fabrication issues of sub-wavelength apertures (<200 nm), the first demonstration
of this type of SNOM by Ash and Nicolls in 1972 [49] used radio waves that allowed
them to use a much large aperture. Using visible light, SNOM was demonstrated
almost simultaneously by Lewis et al. [50] and Pohl et al. [48] in 1984.
The obvious drawback of improving the measurement resolution using this ap-
proach is the loss of optical sensitivity. The light transmission is related to the
aperture size a4 [42]. This means that the sensitivity drops quickly with improved
resolution. In order to enhance the optical sensitivity, often a special metal coating is
added to the probe to improve the damage threshold, which allows for higher powers
to be used. In addition, a larger cone angle of the tip enhances the light throughput
as well. Typical cone angles for etched fiber probes are 40°, resulting in a transmission
of 10-3 - 10-5 for a 50 to 100 nm aperture [42].
2.4.3 Propagation through a sub-wavelength aperture
To understand the properties of a sub-wavelength aperture, a SNOM probe can be
modeled as a small hole in an infinitely thin metal screen. This model was well
explained by Hecht et al. [42], who showed the behavior of a plain wave with
wavenumber k0 = 2π/λ that illuminates the screen with aperture a. The electric
field distribution behind the aperture can be obtained by convolution of the incident
field with the Fourier transform of the aperture. The field amplitude γ(k||, kz) can
then be decomposed in a transverse k|| and longitudinal component kz. For small
apertures (a < λ), kz becomes imaginary and does not propagate anymore.
2.4.4 Practical fiber probes
A small aperture can be created at the tip of an optical fiber to obtain an optical near-
field probe. The aperture size determines the maximum spatial resolution, but also
the light throughput. In probe fabrication one has to compromise between resolution
and sensitivity. Two main fabrication methods are commonly utilized: the heat-and-
pull technique [51] and chemical etching [52–57]. Chemical etching is nowadays the
most popular method because it provides larger cone angles and therefore better
transmission without losing resolution.
In the case of a pulled fiber, the tapered region is often long and the cone angle
small. When the fiber core becomes smaller, interactions with the metal coating
become important and the system can no longer be modeled as a dielectric, but
should be treated as a hollow metal waveguide filled with a dielectric. Novotny and
Hafner [58] calculated the light guidance by metal coated fibers as a function of the
core diameter. As illustrated in Fig. 2.6, when the core diameter decreases the modes
21
2 Introduction to scanning near-field microscopy
2
aluminum
d=250 nm d=160 nmevanescentdecay
HE
11
Figure 2.6: Schematic representation of light propagation in a fiber tip at a wavelengthof 488 nm. Adapted from [42].
run into cutoff one after the other until only the HE11 mode remains and finally also
becomes evanescent at diameters smaller than 160 nm (for λ = 488 nm). Because
the evanescent field decays exponentially towards the tip apex, the light throughput
benefits from a cutoff close to the extremity and thus from a large cone angle as
demonstrated by Novotny et al. [59].
For chemically etched fiber probes the core diameter is not affected by the etching
process and therefore the modes reach the cutoff diameter close to the extremity.
Light transmission is often measured in the far-field by comparing the input power
with the amount of light exiting the tip. For apertures around 100 nm and large cone
angles of 40° - 60° the transmission can be as large as 10−3, rapidly decreasing with
decreasing apertures and cone angles.
2.4.5 Cantilever probes
In a different approach, standard AFM cantilevers have been modified for SNOM
measurements [60–64]. These modified probes have a small aperture at the tip apex,
that can be obtained by focussed ion beam (FIB) milling for example. Using a normal
objective, mounted above the cantilever, light is focused at the tip in order to create
a near-field light source. The advantage of this approach is that much larger cone
angles can be achieved and thus a better optical transmission, typically a factor of
10 to 100 compared to standard etched fiber probes [65]. The fabrication procedure
22
2.4 Scanning near-field optical microscopy (SNOM)
2
is similar to that of normal silicon cantilevers, although more steps are required to
create the hole and make it heat resistant. A detailed description of the fabrication
procedure can, for example, be found in Minh et al. [62].
2.4.6 Scanning feedback mechanism
Different mechanisms for scanning a SNOM probe over a sample are being used.
When a modified AFM type cantilever is used, all AFM operation modes as described
in section 2.2 are available. However, often special fiber probes are used that require
a different system. A fiber probe is glued to a quartz tuning fork that oscillates at its
resonance frequency, generally 32 KHz. A change in the resonance frequency indicates
the approaching sample surface. Two versions of this technique can be used, tapping
mode or shear force mode. In tapping mode the probe oscillates perpendicular to the
sample [66], while in shear force mode the probe oscillates laterally [67].
A standard SNOM fiber probe can also be bent in the form of a cantilever and
accordingly access all standard AFM operation modes [68, 69].
2.4.7 Applications
Aperture SNOM is a method to enable optical imaging beyond the diffraction limit.
In light emitting devices, such as semi-conductor lasers, information about the optical
performance can be obtained as well. An example is shown in chapter 6. Combined
with spectroscopic methods such as fluorescence or Raman spectroscopy, chemical
selectivity can be added.
2.4.7.1 Fluorescence SNOM
Low light throughput requires high sensitivity techniques. The area of cell biology
is especially of interest for SNOM techniques because the dimensions of objects are
just beyond accessible range of confocal microscopy. SNOM can extend the optical
resolution to below 32 nm [70]. It has been shown that SNOM can be a useful method
to image cell membrane processes [71–75]. The advantage of fluorescence is the high
spectroscopic cross section that enables easy combination with optically inefficient
techniques such as SNOM. However, not all biomolecules auto-fluoresce and therefore
special labeling maybe needed. It should be noted that new developments in other
fluorescent super-resolution techniques, such as STED, STORM and PALM [76],
could make SNOM superfluous for biological samples.
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2 Introduction to scanning near-field microscopy
2
2.4.7.2 Raman SNOM
Raman spectra contain valuable chemical information without the need for special
labeling. However the Raman cross section is generally very low. Therefore SNOM
Raman is often combined with surface- or resonance enhanced techniques to acquire
enough signal [77, 78]. Due to these limitations the technology did not develop much
further than proof of principle experiments using molecules with very high scattering
cross sections [65, 69, 79–84].
2.4.7.3 Other applications
SNOM can also be used to detect emitted light in applications where other techniques
are prone to failure. Some examples are the detection of evanescent fields [85, 86],
direct imaging of optical waveguides [87] or measuring the output pattern of small
semiconducting lasers [66]. An overview of literature about applications of aperture
SNOM can be found in Table 2.1.
Table 2.1: Application areas of aperture SNOM
Field Raman Fluorescence Emission / TransmissionBiology [77] [70–72, 74, 75]Physics [66, 87–90]Chemistry [65, 69, 78–84, 91] [92]
2.5 Conclusions
Aperture scanning near-field optical microscopy, which combines the high-resolution
imaging of AFM with optical surface analysis has developed into a versatile research
tool over the last four decades. Biology is the main field where this technique has been
applied because the dimensions of the interesting objects are in the accessible range
of about 30 to 200 nm. Other super-resolution techniques such as STED, STORM
and PALM have gained momentum over the last years [76]. These techniques are
now preferred because of the much higher imaging speed and lack of complication
by tip issues as degradation and sample damage. However, they do not offer the
nanoscale topology offered by SNOM.
Combining aperture SNOM with vibrational spectroscopy, such as Raman, to
access the molecular properties of a sample, is difficult because of the low light
throughput and low spectroscopic cross section. Apertureless SNOM, such as TERS,
on the other hand, solves some of the limitation and can be used to gather high-
resolution molecular information [37, 93–95]. This technique profits from higher
24
2.5 Conclusions
2
sensitivity, resolution and direct molecular information. Applications can be found in
catalysis, molecular biology and chemistry [35, 96–102].
However, in some situations, aperture SNOM might be more appropriate, since
the mechanism is often more reliable, tip degradation or pollution is less critical and
it can be used to directly measure optical emission. This might be beneficial when
measuring semi-conductor laser output or evanescent fields.
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2 Introduction to scanning near-field microscopy
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3Fiber-top sensorsCHAPTER 3