DOI 10.1515/nanoph-2014-0008 Nanophotonics 2014; 3(1-2): 117–124
Review article
Shirly Berezina, Basanth S. Kalanoora, Hesham Taha, Yuval Garini and Yaakov R. Tischler*
Multiprobe NSOM fluorescence
Abstract: In this paper, we demonstrate simultaneous
AFM/NSOM using a dual-tip normal tuning-fork based
scanning probe microscope. By scanning two SPM probes
simultaneously, one dedicated for AFM with a standard
tip diameter of 20 nm, and the second having a 150 nm
aperture NSOM fiber with 200 nm thick gold coating, we
combine the benefits of ∼20 nm spatial resolution from
the AFM tip with the spectral information of a near-field
optical probe. The combination of simultaneous dual-tip
scanning enables us to decouple the requirements for
high resolution topography and probe functionality. Our
method represents a marked shift from previous applications of multi-probe SPM where essentially a pump-probe
methodology is implemented in which one tip scans the
area around the second. As a model system, we apply
dual-tip AFM/NSOM scanning to a sample of spin-cast
nano-clustered Lumogen dyes, which show remarkable
brightness and photochemical stability. We observe morphology features with a resolution of 20 nm, and a nearfield optical resolution of 150 nm, validating our approach.
Keywords: multiprobe; NSOM; SPM; lumogen; FRET;
tuning fork probe.
Shirly Berezin and Basanth S. Kalanoor contributed equally to this
work
*Corresponding author: Yaakov R. Tischler, Bar-Ilan Institute of
Nanotechnology and Advanced Materials, Department of Chemistry,
Bar-Ilan University, Ramat-Gan, 52900, Israel, e-mail: [email protected]
Shirly Berezin and Yuval Garini: Bar-Ilan Institute of
Nanotechnology and Advanced Materials, and Department
of Physics, Bar-Ilan University, Ramat-Gan, 52900, Israel
Basanth S. Kalanoor: Bar-Ilan Institute of Nanotechnology and
Advanced Materials, and Department of Chemistry, Bar-Ilan University,
Ramat-Gan, 52900, Israel
Hesham Taha: Nanonics Imaging Ltd., Jerusalem, 97775, Israel
a
Edited by Aaron Lewis
1 Introduction
Atomic force microscopy (AFM) since its introduction in
1986 [1] has been limited in its technology to single probes
investigating a sample surface. Nonetheless, the goal of
achieving multiprobe AFM operation was a most desirable objective which was difficult to achieve. This article
describes the application of a highly developed multiprobe platform and one of its applications to near-field
scanning optical microscopy (NSOM). In this particular
application, we demonstrate that such a platform can
readily address the dichotomy between achieving the best
NSOM resolution while not sacrificing on the on-line AFM
resolution of the structure.
The development of mulitiprobe scanning probe
microscopy (SPM) technology based on AFM was quite
complex. This complexity arose from a complicated interaction between probe technology, feedback technology,
and the geometry of the construction of the SPM.
The history of such multiprobe technology was therefore initially based on scanning tunneling microscopy and
the guiding of the probes to relatively close proximity was
defined by an on-line scanning electron microscope. There
are several reports on the development of multiprobe STM
equipped with up to four probes [2–9]. The first ambient
example of a two probe STM however did not occur until
2007 [10].
Nonetheless, such technological advances are less
than general due to the fact that STM could not be used
on non-conducting surfaces and without some alternate
methodology, such as SEM or actually performing a scan
with each probe, one could not rapidly place two probes
together. Both of these issues were addressed in a paper
published in 2009 [11].
To put this report in perspective, it is appropriate to
note that Roco et al. [12] in their report in nanotechnology
developments in 2020 emphasized multiprobe SPM as an
important technological advancement placed along with
aberration correction transmissioin electron microcopy,
electro holography, energy dispersive X-ray, etc. This perspective also noted that novel methods to measure electrical, optical, and magnetic properties at the nanoscale
(including multi-probe SPMs) were needed to cause a paradigm shift that would bring invaluable advancement in a
wide variety of technological fields.
The 2009 paper noted above was the first paper based
on a patent originally filed in 2001 [13]. This technology
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118 S. Berezin et al.: Multiprobe NSOM fluorescence
addressed all of the outstanding aspects of how one would
build such a multiprobe SPM based on AFM technology.
Furthermore, the platform could be used not only for AFM
imaging but for the broad spectrum of multifunctional
imaging methodologies that include AFM feedback. It is
for such functional imaging that multiprobe technology
has its greatest potential.
In fact in this first paper by Dallapiccola et al. [11], the
developed multiprobe platform was applied for pumpprobe near-field optical imaging of a plasmonic waveguide in which the propagation of a plasmonic wave was
measured. In this particular experiment, one NSOM probe
was used as a point source of illumination that provided
all “k” vectors to effectively excite plasmons at any point
in the waveguide and the second NSOM probe was used
for collecting the light from the propagating plasmons in
the near-field. A 1/e decay length of 340 nm was measured.
It was already seen in this 2009 study that in a multiprobe system such as this, one probe could image another
probe. This has since been supported by the work of Klein
et al. [14] who has demonstrated with the same platform model, MultiView 4000 Multiprobe SPM (Nanonics
Imaging Ltd, Jersusalem, Israel), that one probe’s feeback
is extremely sensitive to an additional probe’s nanometric
presence. This makes it possible to bring multiple probes
rapidly together and even into contact.
These advances arose from three inter-related developments. The first is the development of a series of functional probes that have probe geometries that permit
probe tips to be in touching distance while keeping the
optical axis completely clear from above and below the
platform. A video of such an operation can be seen on the
internet where a gold nanoparticle probe comes within
the near-field of an NSOM probe and changes the boundary conditions of the emission from the NSOM probe as the
gold nanoparticle approaches (http://www.youtube.com/
watch?v=xnFAW8AXnbY). The second is the development
of ultrathin scanners that can achieve large ranges in X,
Y and Z up to 100 microns and permits new open architecture geometries of scanned probe microscopes [15]. The
third is the development of normal force tuning-fork feedback which shows that the ultimate in force sensitivity
with glass based multiprobes enabled probes for NSOM,
electrical, thermal, plasmonic, ion conductance, fountain-pen nanolithography, and scanning electrochemical microscopy applications (http://www.nanonics.co.il/
products/spm-probes-and-nanotools.html). For example
it has been shown that with such probes and the platform used in the present study, a force sensitivity of 1.6 pN
could be achieved, which is more sensitive than any other
AFM based feedback technique developed thus far [16].
A major challenge that is pervasive in nearly all functional applications that include SPM is that the functional
probes associated with these applications do not always
achieve the ultimate in AFM topographic resolution. This
is due to the additional dimensions inherent in some
functional probes that are based on AFM feedback. The
example that we focus on in this paper is nano-optical
measurements with NSOM. Since the inception of NSOM,
this has been a problem that has limited the AFM resolution of an NSOM probe due to the coating around the
active area that defines the near-field optical element.
In this paper we concentrate on fluorescence imaging
with NSOM. For such imaging, the best choice is aperture
based NSOM. This is due to the fact that fluorescence is
very sensitive to bleaching and thus, in aperture based
NSOM only the pixel that is being interrogated is bleached.
Fluorescence however requires maximal photon flux from
the nano-aperture of an NSOM probe. For such maximal
photon flux, the cone angle of the probe has to be large
and such cone angles compromise the AFM resolution
which is defined by the outer diameter of the probe and
not the aperture which defines the NSOM resolution.
Thus, multiprobe technology permits one the potential
to achieve maximal AFM and NSOM resolution on line for
excellent structural and optical nano-characterization.
In addition, without the best AFM resolution one
often requires the NSOM probe itself to be used to search
for potential fluorescence structures. However, this in
itself compromises one of the advantages of fluorescence
NSOM where a virgin pixel not exposed to light can be
interrogated with this singular nano-optical technique
which confines light nanometrically in X, Y and Z unlike
any other optical technique known.
All of the above problems, which have been associated with NSOM fluorescence since its inception [17], can
be overcome by using the multiprobe technology that has
been applied to fluorescence NSOM imaging for the first
time in this paper. Specifically, the main concept introduced here is the use of a dual probe. The first probe can
image the surface at the highest AFM resolution to locate
the position of particles with potential fluorescent spectral properties. The second is chosen to give the highest
NSOM signal. Thus, this can involve making a relatively
large cone angle probe which reduces the length of the
transport of light in the tapered region of the NSOM fiber
probe which has a diameter that is below the cut-off frequency of the optical fiber where light cannot propagate
without loss in this metal coated region. Furthermore,
such a probe which generally has a large force constant
in the 1–10 N/m range can readily be used to scan soft
surfaces such as the materials studied in this paper since
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S. Berezin et al.: Multiprobe NSOM fluorescence 119
the probe is complexed to a tuning fork for normal force
tuning fork force sensitivity.
As a model system, we investigate dual tip AFM/
NSOM scanning on thin films of Lumogen dyes. Lumogen
dyes, manufactured by BASF, are good initial examples
since they exhibit high fluorescence quantum yield and
relative photochemical stability even in air. This allowed
in this initial study multiple scans with the NSOM probe to
verify our conclusions.
The dye Lumogen Red that was used is reported
to have a quantum yield exceeding 90%, and the dye
Lumogen Orange is characterized by similar performance.
Thin films of Lumogen doped into polymeric host-matrices
are being implemented as solar concentrator layers due to
their ability to withstand high intensity solar excitation
for very long periods of time and are also used in thin film
organic waveguide laser architectures [18, 19].
Thus, these dyes permit scans of long duration for the
highest signal to noise in scanning optical measurements.
Therefore, Lumogen dye is an attractive candidate material to investigate.
Prior to our work, to the best of our knowledge, these
dyes have not been reported in the NSOM/SNOM literature. Perhaps the reason is that their fluorescence is
severely quenched when a neat film is prepared as occurs
for perylene and other laser dyes [20]. Other forms of perylene diimide have been studied by NSOM [21] but not
for near-field fluorescence [22], likely due to pronounced
“concentration quenching”.
In order to obtain the high brightness of the Lumogen
dyes even in solid state, we prepared composite films of
Lumogen Red doped into a Lumogen Orange host matrix.
In the doped layer, the spacing between Lumogen Red
molecules is sufficient to prevent concentration quenching of the fluorescence. Using Lumogen Orange as the
host introduces the possibility of FRET to occur between
the host and dopant dye molecules, due to the good spectral overlap between Lumogen Orange’s fluorescence and
Lumogen Red’s absorption. FRET sensitizes the fluorescence of the acceptor molecule and thus enables a much
larger fraction of laser light to be absorbed and transferred
to a smaller fraction of Lumogen Red molecules. Lumogen
Orange also has stronger absorption of the 532 nm light
used for the near-field excitation. On glass and silicon
substrates, for specific preparation conditions, the dye
molecules can pool into sub- micron scale islands that are
suitable for dual-tip AFM/NSOM SPM characterization.
2 Experimental section
Samples of nano-clustered Lumogen dyes were probed
using AFM, NSOM, and simultaneous dual-tip AFM/NSOM
following the schematic of Figure 1A,B. All measurements
were performed using a dual microscope from Nanonics Imaging which was free-space coupled to a LabRam
HR Micro-Raman microscope from Horiba Jobin Yvon as
shown in Figure 1B.
Upon initial examination with white-light far-field
illumination, samples were scanned using standard AFM
tuning fork probes. Gain and feedback parameters were
adjusted to obtain high resolution images. Fluorescence
of the sample was mapped via far-field excitation and
LabRAM
A
B
Laser
Optical fiber
AFM
NSOM
Sample with
scanning stage
Microscope
objective
APD
Figure 1 Schematic of dual-tip SPM system and the experiment. (A) Microscope image of approaching AFM and NSOM tuning fork probes.
(B) Experimental set-up consisting near-field NSOM probe with 150 nm aperture illuminated with 532 nm fiber coupled laser, AFM probe
with 20 nm diameter, and bottom microscope with 50 × objective and aligned APD for light collection.
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120 S. Berezin et al.: Multiprobe NSOM fluorescence
For these initial steps in multiprobe fluorescence
NSOM described in this paper, the sample was excited
with a 532 nm laser in the near-field through an NSOM
fiber probe with 150 nm aperture. The fluorescence was
collected in transmission mode through a long working
distance 50 × objective (N.A. = 0.45, W.D. = 17 mm) of the
inverted microscope from below. The fluorescence intensity was detected in transmission mode with an APD from
Excelitas (SPCM-AQRH-14) after filtering out the laser excitation using a Semrock 532 nm Razor Edge long pass filter
(LP03-532RE-25).
emission using the LabRam coupled to the multiprobe SPM
system in order to obtain on-line micro-PL spectra. Then,
NSOM was performed using near-field excitation and a
photon-counting avalanche photo-diode (APD) to collect
the near-field excited fluorescence through the objective
of the microscope. From the NSOM scan, we obtained the
near-field mapping of fluorescence and information about
topography. Finally, the AFM tip was brought into feedback with the sample and then into soft-contact with the
NSOM probe (See Figure 1A). The details of achieving softcontact are described below. With both probes in feedback
and soft-contact with each other, the sample was scanned
to obtain dual-tip AFM/NSOM SPM images. A closed loop
scanner from Physik Instruments (PI) was used to perform
substrate-scanning of the samples.
2.2 Sample preparation
Lumogen Red (BASF Lumogen F Red 305) and Lumogen
Orange (BASF Lumogen F Orange 240) were obtained
from BASF free of charge and used without further purification. Chemical structures of the two lumophores are
shown in Figure 2A,B respectively. Solutions for doped
layers of Lumogen consisted of 95% Lumogen Orange
and 5% Lumogen Red. First, 9.5 mg of Lumogen Orange
and 0.5 mg of Lumogen Red were combined and then dissolved in 10 ml of acetone to obtain a solution with net
concentration of 1.0 mg/ml. Then the solutions were kept
stirring for 3 h in order to dissolve the dye completely in
the solvent.
2.1 System design
The experimental setup consists of a commercial scanning probe microscope (SPM) system Hydra Multi View
4000 (Nanonics Imaging Ltd, Jerusalem, Israel). The Multi
View 4000 consists of a dual microscope to provide optics
for both upright and inverted microscopy based upon two
Olympus BXFM microscopes. This configuration allows for
exciting the sample using transmission mode far-field and
near-field optics and also reflection mode if so desired.
A
C
O
O
N
2500
Lumogen Orange+Red
Lumogen Red
Lumogen Orange
N
2000
Lumogen Orange(LO)
B
O
O O
N
O
1500
1000
O
N
O O
Intensity (a.u)
O
O
O
500
0
550
600
650
700
750
800
850
900
Wavelength (nm)
Lumogen Red (LR)
Figure 2 Chemical structure and corresponding fluorescence spectra of Lumogen dyes under study. (A) Molecular structure of Lumogen
Orange, (B) Lumogen Red, and (C) fluorescence spectra of Lumogen Orange (open circles), Lumogen Red (thick line) and the doped film of
Lumogen Orange and Red, 19:1 (w/w) shown in (filled squares).
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S. Berezin et al.: Multiprobe NSOM fluorescence 121
Lumogen films were prepared on a glass substrate
of sizes 25 × 25 mm2 using spin coating method. The substrates are cleaned via sonication in Micro-90 semiconductor grade detergent, de-ionized water, and acetone, and
then rinsed in boiling isopropyl alcohol and dried under a
stream of nitrogen. Immediately following cleaning, substrates were exposed to oxygen plasma for 120 s. Lumogen
layers were prepared by adding sufficient quantity of solution to cover the substrate surface. Then the substrate was
rotated at 1000 rpm for 90 s with an acceleration of 1000
rpm/s, using a Headway Research PWM32 spin-coater.
3 Results
Figure 2C shows the fluorescence spectra of Lumogen
Orange, Lumogen Red, and the doped film, from upon
excitation with a laser at 532 nm. The Lumogen Orange
sample has a photoluminescence peak (PL) at a wavelength of 622 nm, and the PL peak for the Lumogen Red
sample is at 609 nm. The doped film shows two peaks; the
major peak corresponds to Lumogen Red (628 nm), and
the small satellite peak is from Lumogen Orange (542 nm).
The intensity of the doped film compared to the neat film
of Lumogen Red shows a dramatic increase in PL intensity. This is suggestive of FRET sensitized fluorescence
of the acceptor fluorophore. The acceptor Lumogen Red
is only 5% of the film concentration, and yet it shows 2.3
times more fluorescence at the PL peak compared to the
neat Lumogen Red sample. At the same time, since in the
-25
0.0
A
160
B
0.5
-20
140
1.0
-15
Y (µm)
doped film, the Lumogen red molecules are further apart,
the quantum yield is expected to be higher than in a neat
film even without FRET being operative. While the mechanism of fluorescence enhancement still needs to be investigated, the doped film clearly provides greater PL signal
than either of the neat films, and therefore it was chosen
for the investigations of dual-tip AFM/NSOM scanning.
Figure 3A shows the microscope image of the sample
using far-field white-light illumination. The Lumogen
molecules cluster into sub-micron sized islands. F
­ ar-field
micro-PL mapping of the fluorescence is shown in
Figure 3B. The integration time was set to 250 ms per
point, and a 100 × 0.9 NA objective was used, with a 532 nm
wavelength laser excitation. Based on these images, the
spatial profile of a typical island is on the order of 1 μm.
AFM scans of the sample show a much higher resolution
image of the morphology, indicating the actual profile of
such features is 600 nm in lateral extent or less depending
on the island chosen.
After performing baseline AFM-only and NSOMonly measurements, dual-tip AFM/NSOM simultaneous
scanning was implemented. Figure 4A–C show the topo­
graphy obtained from the NSOM probe and AFM probe,
and the simultaneously obtained near-field fluorescence
image. Figure 4F is the height profile from the NSOM
probe, which shows good correlation between maximum
height and maximum fluorescence, but poor lateral topographical resolution. For the same features, the simultaneous AFM scan shows much better lateral resolution
(See Figure 4D) revealing sub-140 nm diameter islands on
the film. The near-field fluorescence of the same trace is
1.5
120
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2.0
100
-5
2.5
0
3.0
5
3.5
60
10
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40
80
4.5
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20
5.0
20
0
5.5
25
6.0
-20
-10
0
X (µm)
10
20
-20
0
2
4
6
Figure 3 Far-Field images of doped Lumogen sample. (A) White-light image from reflection of the sample. (B) Micro-PL mapping using a
532 nm laser as the source of excitation.
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122 S. Berezin et al.: Multiprobe NSOM fluorescence
A
B
D
E
70
F
5
60
4
40
30
20
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Z (nm)
Z (MHz)
50
Z (nm)
C
2
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10
0
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5
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80
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X (µm)
5
6
Figure 4 Dual-Tip AFM/NSOM SPM results for thin film of Lumogen Red doped into a Lumogen host matrix (5%/95%). (A) Topography
obtained from AFM tip in soft-contact with NSOM probe, (B) simultaneously measured near-field fluorescence signal obtained from NSOM
probe showing spatial heterogeneity of the NSOM fluorescence in the image, (C) Topography obtained from NSOM probe, (D) Horizontal
profiles of AFM topography, (E) NSOM tip excited fluorescence signal, and (F) NSOM topography.
shown in Figure 4E. The spatial extent of the nano-islands
obtained from the optical signal is comparable to the AFM
topography with the AFM probe, indicating the complementarity between AFM with an AFM probe and the associated NSOM probe fluorescence images. This correlation
is far superior to anything that could be discerned with the
AFM resulting from the convolution of the NSOM probe tip
diameter (550 nm) with the sample. This is even though
the NSOM tip by itself has a core diameter of 150 nm.
Due to the robustness of the Lumogen dyes, the
sample showed negligible photo-bleaching during the
measurement. Thus, for these initial steps in multiprobe
NSOM fluorescence, it allowed for multiple measurements
to verify fully this new direction in fluorescence NSOM. In
particular, we were able to scan the same location more
than 10 times without any observable decrease in PL
signal.
4 Discussion
From these first demonstrations, we can surmise that
simultaneous dual-tip AFM/NSOM SPM appears to be a
general method for obtaining high-spatial resolution AFM
topography combined with the spectral functionality of
an NSOM probe. There seems to be every indication that
as a methodology, dual-scanning can be applied to many
other nano-optical modalities including AFM/TERS and,
in addition, a wide variety of hyphenated AFM/SPM techniques in order to decouple the requirements of obtaining
high-resolution AFM together with additional SPM functionalities. Although these conclusions were a result of
our use of the Nanonics MultiView 4000 Multiprobe SPM
platform, we believe these conclusions should generally
apply to any future multiprobe system based on the principles of the platform we have used in this paper.
Figure 5 shows the three-dimensional image of the
AFM topographical map overlapped with the NSOM fluorescence, and the good correlation between the two simultaneously obtained scans. In performing the dual-tip SPM
scanning, we noticed that there is an offset between the
two images of about 0.5 μm. This offset means that as
the fluorescent material is scanned below the dual-tips,
each tip senses the same morphology at a slightly different sample scanner position. This is entirely reasonable
because the two tips have some finite lateral extent and
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S. Berezin et al.: Multiprobe NSOM fluorescence 123
In summary, the data shown in Figure 4 clearly
show that the NSOM resolution closely follows the AFM
resolution obtained with the AFM probe. Using this
comparison, the interpretation of the NSOM relative to
the structural characteristics of the sample are readily
delineated while a similar delineation of even these
fairly large structures is difficult to unravel with the AFM
from the NSOM probe. Thus, this indicates the potential
of enhancing fluorescence NSOM to a level that adds
excellence of structural correlation that is critical in a
wide variety of fields from fluorescent nanoparticles to
biological membranes. This level of structural correlation has the potential to lead to new horizons in such
imaging.
Figure 5 Three-dimensional image of dual-tip AFM/NSOM SPM
results. AFM topography is overlaid with NSOM fluorescence map,
and shows good correlation between the two simultaneously
obtained images.
the points of contact with the sample for each tip are not
necessarily the points of the tips that are in contact with
one another.
On a technical note to carry out such dual-tip SPM
measurements, it is necessary to achieve “soft-contact”
between the AFM tip and the NSOM probe. First, each tip
is brought into feedback with the sample. Then, while
both tips are still in feedback, they are brought together
into soft-contact using piezo-positioners. The Nanonics
Multi-View 4000 is especially suited for landing both tips
together because the system allows for off-setting, i.e.,
positioning, one of or more tips using piezo-positioners,
and has a tip-scanning mode of operation which can
readily be switched to sample scanning. Furthermore,
the system can be configured so that feedback is maintained via the tip-scanner, even though the substrate is
being rastered during the measurement. From the piezocontroller, one observes the onset of soft-contact by noticing an alteration in the z-channel piezo-voltage, at which
point the dual-tip scan can be initiated. Once soft contact
was achieved, any offset in relative tip positions of the
two probes could be readily adjusted since the probes
were continually maintaining feedback. This is especially
due to the fact that the controller offsets permit lateral
adjustments even while maintaining tapping mode feedback without lateral force. Finally, based on the sensitivity of the probes to each other’s presence, it is feasible to
expect that future developments will include automatic
algorithms for closely fixing the relative probe positions.
5 Conclusion
We have shown that simultaneous dual-tip AFM/NSOM
scanning combines the benefits of high spatial resolution
morphological imaging with near field spectral information. As a model system, we chose to implement dual-tip
simultaneous SPM on spin-cast Lumogen dye nano-clusters. The clusters were composed of donor and acceptor
molecules, Lumogen Orange and Lumogen Red respectively, to achieve high absorption of the NSOM excitation
and yet high brightness. This work is the first report of
NSOM on Lumogen dyes, and the first demonstration of
simultaneous dual-tip scanning, fluorescent NSOM. We
are confident that the results of this study should lead to
many exciting future applications.
Acknowledgments: The authors would like to thank Sivan
Harazi for providing the initial samples that triggered the
need and the idea for dual-tip AFM/NSOM SPM, BASF
Chemical Corporation for giving us the Lumogen Red and
Orange free of charge, and Alex Palatnik for contacting
BASF and arranging the delivery of the dye, Dr. Hagay
Shpaisman for lending us a 5.0 MP camera, and Prof.
Aaron Lewis for helpful discussions. The authors also
gratefully acknowledge their sources of funding: the
Israel Science Foundation for ISF-Individual and ISFInstitutional Equipment Grants. S. B. and B.S.K. would
like to thank Bar-Ilan University for providing scholarship
and fellowship respectively.
Received March 17, 2014; accepted March 17, 2014
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124 S. Berezin et al.: Multiprobe NSOM fluorescence
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