APPLIED PHYSICS LETTERS 104, 121906 (2014)
Highly confined, enhanced surface fluorescence imaging with
two-dimensional silver nanoparticle sheets
Eiji Usukura,1 Shuhei Shinohara,1 Koichi Okamoto,1 Jaehoon Lim,2 Kookheon Char,2
and Kaoru Tamada1,a)
1
Institute for Materials Chemistry and Engineering, Kyushu University, Fukuoka 812-8581, Japan
The National Creative Research Center for Intelligent Hybrid, School of Chemical and Biological
Engineering, Seoul National University, Seoul 151-744, South Korea
2
(Received 10 February 2014; accepted 11 March 2014; published online 25 March 2014)
A method of obtaining highly confined, enhanced surface fluorescence imaging is proposed using
two-dimensional (2D) silver nanoparticle (AgMy) sheets. This technique is based on the localized
surface plasmon resonance excited homogeneously on a 2D silver nanoparticle sheet. The AgMy
sheets are fabricated at the air–water interface by self-assembly and transferred onto hydrophobic
glass substrates. These sheets can enhance the fluorescence only when the excitation wavelength
overlaps with the plasmon resonance wavelength. To confirm the validity of this technique, two
separate test experiments are performed. One is the epifluorescence microscope imaging of a
quantum dot 2D sheet on the AgMy 2D sheet with a SiO2 spacer layer, where the fluorescence is
maximized with the 20 nm SiO2 layer, determined by the F€orster resonance energy transfer
distances. The second experiment is the imaging of a single fluorescence bead with a total internal
reflection fluorescent microscope. We confirmed that the AgMy sheet provides a 4-fold increase in
fluorescence with a 160-nm spatial resolution at 30 ms/frame snapshot. The AgMy sheet will be
a powerful tool for high sensitivity and high-resolution real time bioimaging at nanointerfaces.
C 2014 AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4869560]
V
Surface plasmon resonance (SPR) sensors were developed for the real-time detection of bio-related molecules on
surfaces about 30 years ago.1 Conventional SPR sensors
detect refractive index change at the metal interface, with the
advantage of label-free detection. However, the sensitivity of
this technique was not as high as in fluorescence detection
and was one of the reasons why SPR has never dominated
fluorescence biosensing in the past decade. Recently, surface
plasmon fluorescence spectroscopy (SPFS) has been developed to offer higher sensitivity than a conventional fluorescence system.2 Here, propagating SPR excited on a metal
thin film is used to enhance the fluorescence signal.
The electric field excited by the propagating SPR is
known to be 20–100 times stronger compared with that of
the incident light, and the penetration depth is a few hundred
nm from the interface. Thus, the SPFS technique has been
applied for various high-sensitive biosensors at the interface,
especially to challenge their detection limit.3 However, the
weak point of the propagating SPR was the spatial resolution
caused by their propagating property of more than a few lm
at the interface, which has not allowed high-resolution molecular imaging.
In this study, we propose to utilize localized surface
plasmon resonance (LSPR) instead of the propagating SPR
for highly sensitive and high-resolution fluorescence imaging. The LSPR excited on noble metal nanoparticles has the
potential to excite even stronger electric fields than the propagation mode, especially at the nano-gaps between adjacent
particles.4 The primary advantage of LSPR is its spatial
confinement (both in plane and out), which makes highresolution imaging possible. On the other hand, one potential
a)
Electronic mail: tamada@ms.ifoc.kyushu-u.ac.jp
0003-6951/2014/104(12)/121906/5/$30.00
problem is the difficulty of controlling the spatial distribution
of the particle arrays on the surface. The top-down technology creates regular arrays; however, the density of the arrays
is limited (the distance between the arrays is typically
100 nm or more) with current technology.5 The bottom-up
technology provides nano-sized particles immobilized on
solid substrate; however, these are mostly randomly
located.4
In a previous study, we have succeeded in fabricating
Ag-nanoparticle, two-dimensional (2D) crystalline sheets by
self-assembly at the air–water interface. Fig. 1 shows a scanning electron microscope (SEM) image of 2D sheet composed of myristate-capped Ag nanoparticles (AgMy)
transferred to hydrophobic Si-wafer. The AgMy were synthesized by thermal reduction of silver acetate precursor in
the melt of myristic acid as described before.6 In such a
sheet, the LSPR wavelength can be tuned by the kind of
metal species (Ag or Au), the size of particles, the distance
between adjacent particles controlled by organic capping
molecules, and their macroscopic domain size. When these
parameters are in the correct ranges, the sheet can have a
sharp, strong LSPR absorption band. In this report, we utilize
a 2D sheet composed of AgMy for LSPR field–enhanced fluorescence microscope imaging.
In order to confirm the validity of this technique, we performed two separate test experiments. One was epifluorescence microscope imaging of a quantum dot (QD) 2D sheet
on an AgMy sheet with a SiO2 spacer layer. Another was
imaging of a single fluorescence bead (1 lm in diameter)
under a total internal reflection fluorescence (TIRF) microscope. We then compared the intensities and the spatial resolution of the fluorescence images with and without the
AgMy sheet (AgMyþ or AgMy) quantitatively.
104, 121906-1
C 2014 AIP Publishing LLC
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Usukura et al.
FIG. 1. (a) Scanning electron microscope (SEM) image of AgMy nanosheet
(monolayer) on Si wafer. (b) A schematic drawing of interdigited structure
of AgMy.
Fig. 2(a) shows the structure of the test experiment with
epifluorescence microscope (ECLIPSE 80i, Nikon, Japan).
The QDs with oleic acid capping were synthesized as
described in the literature.7 The QD monolayer sheet
was fabricated in the same way as the AgMy sheet by
FIG. 2. (a) A structure of the test experiment with QD sheet using epifluorescence microscope. (b) The absorbance spectrum of the AgMy sheet (gray
line) and excitation (blue line) and fluorescence (red line) spectra of the QD
sheet. (c) The QD fluorescence images on AgMyþ and AgMy with a different thickness of SiO2 spacer layer, 10 nm, 20 nm, 50 nm, and 100 nm, and
the histograms of ‘counts per pixel’ obtained from the fluorescence images.
The black and gray bars represent the data on AgMyþ and AgMy, respectively. The inserted numbers are averaged ‘counts per pixel.’ EF is the
enhancement factor calculated from the averaged ‘count per pixel.’
Appl. Phys. Lett. 104, 121906 (2014)
self-assembly at the air-water interface. The excitation wavelength was 450–490 nm under a filtered mercury lamp, which
overlapped with the LSPR absorption band of the AgMy
sheet (Fig. 2(b)). The epifluorescence images of the QD
sheet on AgMyþ and AgMy substrates taken by a chargecoupled device (CCD) camera (DS-Filc, Nikon), where SiO2
spacer thickness is varied from 10 to 100 nm, are shown in
Fig. 2(c). When QDs layer was deposited directly on the
AgMy sheet, the fluorescence from the QD layer was completely attenuated by F€orster resonance energy transfer
(FRET)8 (data are not shown). The fluorescence was still
slightly lower on AgMyþ than that on AgMy with a 10 nm
SiO2 spacer layer (enhancement factor (EF): 0.93). The
enhanced fluorescence was obtained for the sample with a
20 nm SiO2 layer (EF: 1.18), in which the irradiated area was
homogeneously enhanced. The cracked pattern in the image
originates from the defects of the QD layer. When the thickness of the SiO2 spacer layer was more than 50 nm, the fluorescence intensity became nearly identical between the
AgMyþ and AgMy samples (EF: 0.99). The above phenomenon is reasonably explained by the correlation between
the LSPR field and FRET distances.9 The maximum EF
obtained was only 1.18 since the excitation by the directly
irradiated light from the top surface is dominant in this system (the influence of LSPR field enhancement at the bottom
layer was not so significant).
Next, we tested the efficiency of the AgMy sheet as a
substrate for high-resolution fluorescence imaging under a
TIRF microscope (ECLIPSE Ti, Nikon). Since the TIRF
microscope utilizes an evanescent field, it can have a high
spatial resolution for both in-plane and depth directions,
overcoming the light diffraction limit. However, the absolute
intensity of fluorescence excited by the evanescent light is
much weaker compared with the conventional fluorescence
microscope images taken by ordinary light. It needs
improvement, especially when we consider high-speed realtime imaging. The propagating SPR excited on a metal thin
film seems to be useful for this purpose, too;10 however, as
mentioned above, the propagation mode of the SPR is limited in spatial resolution. We believe that our AgMy sheet
may realize both high sensitivity and high spatial resolution
under TIRF microscope imaging.
Schematics of test samples with a TIRF microscope are
shown in Fig. 3(a). Fig. 3(c) shows the fluorescence snapshot
images of a single fluorescence bead (TransFluoSpheres,
Molecular Probes, 1 lm in diameter) on the AgMyþ and
AgMy substrates, taken by a high-speed CCD camera
(EM-CCD C9100-13, Hamamatsu Photonics). The time resolution was 30 ms/frame. The laser wavelength of 488 nm is
overlapped with the LSPR absorption band of the AgMy
sheet (Fig. 3(b)). The fluorescence intensity of the single
bead was estimated from the photon count per pixel after
background subtraction. The maximum count obtained from
the brightest pixel was 473 on AgMyþ, and was only 112
per pixel on AgMy. We estimated the EF by LSPR to be
4.2. Concerning the spatial resolution, one pixel in the image
is about 160 nm squared determined by the lens magnification and the resolution of CCD camera, which was clearly
resolved at the edge of the bead image on AgMyþ in
Fig. 3(c). The measured diameter of the bead located on
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FIG. 3. (a) A structure of the test experiment with fluorescence beads on
AgMyþ and AgMy using TIRF microscope. The diameter of fluorescence
bead is 1 lm. Incident light wavelengths are 488 nm and 591 nm. (b) The absorbance spectrum of AgMy sheet (gray line) and excitation (blue line) and
fluorescence (red line) spectra of fluorescence beads. (c) Fluorescence snapshots of single beads (30 ms/frame). The left figure was taken on AgMyþ
and the right was on AgMy. The angle of incidence was 74 degrees. The
maximum photon count per pixel was 473 on AgMyþ and 112 on AgMy.
The EF was estimated to be 4.2. The movies are available online
(Multimedia view) [URL: http://dx.doi.org/10.1063/1.4869560.1] [URL:
http://dx.doi.org/10.1063/1.4869560.2].
AgMyþ was about 1 lm. The evanescent depth calculated at
an incident angle of 74 degrees was 64 nm (see the upper
and lower X-axes of Fig. 4 at 488 nm), and the cross sectional diameter of the bead at this depth position was
expected to be 510 nm by a simple geometrical calculation.
It means that the obtained image, 1 lm in diameter, seems to
be slightly enlarged compared with the expected one; however, when we consider the light confinement in the bead
(with a high refractive index), the measured size would be
reasonable. Thus, we could confirm that the TIRF image on
Appl. Phys. Lett. 104, 121906 (2014)
the AgMy sheet keeps the same spatial resolution as that
without the sheet, i.e., no decline in the spatial resolution by
the LSPR field-enhancement was confirmed.
Fig. 4 summarizes the correlation between the EF and
the evanescent light depth for 488 nm and 591 nm wavelengths of lasers. The 488 nm laser overlaps with the LSPR
absorption band of AgMy sheet, while 591 nm laser does not,
as shown in Fig. 3(b). The EF value was calculated from the
total photon counts of the bead (15 15 pixels). The largest
EF value was obtained when the 488 nm laser was employed
with the largest incident angle (76 degrees), i.e., at the shallowest evanescent field. On the other hand, the enhancement
remained quite small for 591 nm wavelength, independent of
the evanescent light depth. The data revealed that the LSPR
field–enhanced factor was indistinct at the absorption edge of
the AgMy sheet. In other words, the enhanced fluorescence
obtained with the 488 nm wavelength was certainly due to the
LSPR field enhancement on the AgMy sheet.
The fluorescence intensity from the bead can be estimated by the local intensity of the evanescent light, the EF
from LSPR, and the quenching by FRET. The intensity of
evanescent light (I) is given by the following single exponential functions (1) and (2). Here d is the distance from the
plane of total reflection (cover glass–water interface), d0 is
the depth of evanescent light, k is the wavelength of incident
laser, h is the incident angle, and n1 and n2 are the refractive
indices of cover glass (1.522) and water (1.33), respectively.
d
;
(1)
I ¼ I0 exp d0
d0 ¼
k
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi :
4p n21 sin2 h n22
(2)
In order to estimate the EF by LSPR, first we conducted
a simulation using the finite-difference time-domain (FDTD)
method with commercial software, Poynting for Optics
(Fujitsu, Japan). Here, the spatial distribution of the electric
field was calculated in a manner similar to our previous
study,6 based on hexagonally packed 5-nm Ag nanoparticles
with the interparticle distance of 2 nm. The particles were
encapsulated into 2-nm thick model surfactant layers (refractive index n ¼ 1.5), and the AgMy model sheet was placed at
the glass–water interface. In the simulation, the pulsed plane
light composed of a differential Gaussian function centered
at 1.5 fs with a width of 0.5 fs, an intensity of 1 V/mm, and a
FIG. 4. Experimentally obtained EFs
plotted against the incident angle and
the evanescent light depth of the excitation light, at the wavelength of
488 nm and 591 nm. The evanescent
light depths are calculated from
microscope-specific equations. Solid
lines are theoretically obtained EF values from the evanescent field intensity
and LSPR and FRET effects as
described in Fig. 5.
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Usukura et al.
Appl. Phys. Lett. 104, 121906 (2014)
bandwidth of around 600 THz (500 nm wavelength), irradiated the AgMy layer from the backside of the glass substrate.
The detection point of the local electric field was located at
the center position of adjacent AgMy particles (“hot spot”)
in the plane of the sheet (Z ¼ 0) and then moved into the
water phase, perpendicular to the surface (Z > 0). A schematic for the FDTD simulation model is given in Fig. 5(a).
Optical intensities at 488 nm and 591 nm wavelengths of
light were calculated from the square of local electric fields,
as shown in Fig. 5(b) with dashed dotted lines, where the
original light intensity was normalized to unity. The calculation result revealed the largely enhanced electric field localized within a 5 nm region from the particle for both
wavelengths. We note that the intensity was 10 times stronger at 488 nm compared with 591 nm.
FRET’s attenuation factor was calculated using below
equations:11,12
"
4 #1
df
Id
¼ 1þ
;
I1
d
"
c3 U d
df ¼ 0:225 2
x xf kf
(3)
#1=4
:
(4)
Here Id is the fluorescence intensity at distance d, I1 is the
fluorescence intensity at infinitely far from the metal surface,
orster distance, c is the speed of light in vacuum, x
df is the F€
is the angular frequency of the donor’s electronic transition,
and Ud is the quantum yield of donor. Moreover, xf is the
Fermi angular frequency of metal while kf is the Fermi wavenumber of metal. Calculated FRET profiles are shown in
Fig. 5(b) with dotted lines. FRET distances df indicate the
separation between the fluorophore and the metal surface.
The typical value of the FRET distance was 5–7 nm.2 In this
FIG. 5. (a) The model for the FDTD calculation of the AgMy 2D nanosheet,
and the spatial distribution of the local electric field calculated from the
model. The diameter of Ag core is 5 nm, and the gap distance is 2 nm. (b)
The calculated intensity of the optical field under the influence of LSPR and
FRET at 488 nm and 591 nm. Dashed dotted lines are the LSPR field intensities calculated by FDTD simulation. Dotted lines represent the FRET
effect given by Eqs. (3) and (4). Solid lines correspond to the relative intensity of the optical field as the products of FRET and LSPR effects.
paper, we used df ¼ 5 nm for subsequent calculations. Solid
lines in Fig. 5(b) correspond to the relative intensity of the
optical field as the distance-dependent products of the FRET
attenuation factors and the LSPR EF. An optical field
enhanced 50-fold field was obtained a few nm from the
plane. Although it was attenuated drastically by distance, a
factor of 10 enhancement remained 10 nm from the plane,
with a long tail extending out to 50 nm. On the other hand,
no enhanced optical field was obtained with 591 nm wavelength of light. The dip of the electric field appearing at 5 nm
from Z ¼ 0 was due to the organic capping layer.
Solid lines in Fig. 4 are the theoretically obtained EF
values from the evanescent field intensity, LSPR, and FRET
effects described in Fig. 5(b). The trends in the theoretical
values agreed with the experimental ones, i.e., the EF value
increased as the incident angle increased at 488 nm while no
enhancement was obtained at 591 nm. The difference
between theoretical and experimental values may be caused
by the optical configuration of the microscope, which was
completely omitted in the theoretical calculation. Possible
effects include light extraction efficiency through the high
refractive index AgMy layer and the objective lens.
Our experimental and theoretical results show that the
LSPR excited by the AgMy sheet strongly confines light few
tens of nanometers from the interface and enhances the fluorescence only when the excitation wavelength overlaps with
the LSPR wavelength. This technique must be certainly
effective for the imaging of real nanointerfaces, requiring
high signal-to-noise ratios, high spatial resolution, and
high speed (the time resolution of this experiment was
30 ms/frame by the performance limitation of the CCD camera; however, it can be certainly faster).
In summary, we demonstrate the LSPR field–enhanced
fluorescence imaging on an AgMy sheet by two separate
experiments: One is the epifluorescence microscope imaging
of a QD 2D sheet, and another is the TIRF microscope imaging with a single fluorescence bead. We have confirmed that
our AgMy sheet enhances fluorescence signals for both
microscope images, especially with the TIRF microscope
under evanescent light, where the EF was 4, with a spatial
resolution of 160 nm at 30 ms/frame snapshot. The homogeneously excited LSPR field on a large surface by selfassembled metallic nanoparticles must be a powerful tool for
highly sensitive and high-resolution imaging of biomolecules
such as single proteins, tissues, and cells, especially for the
dynamics of a single molecule requiring high-speed imaging
(sufficiently strong fluorescence signal with a high signal-tonoise ratio and high spatial resolution are required).13
This work was supported by NEXT Program in JSPS
Funding Program of Japan. K.T. acknowledges Ms. K.
Michioka and Dr. Ikezoe for the SEM observation. K.C.
acknowledges the financial support from the National
Creative Research Initiative Center for Intelligent Hybrids
(No. 2010-0018290) through the National Research
Foundation of Korea (NRF) grants and the BK Plus Program
funded by MEST of Korea.
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