In Vitro and In Vivo Nonlinear Optical
Imaging of Silicon Nanowires
Yookyung Jung,†,|Ling Tong,‡,|Asama Tanaudommongkon,‡Ji-Xin Cheng,‡,§
and Chen Yang*,†,‡
Department of Physics, Department of Chemistry, Weldon School of Biomedical
Engineering, Purdue UniVersity, West Lafayette, Indiana
Received April 9, 2009
fromdimension-controllable silicon nanowires as small as 5 nmin diameter. The nonlinear optical signals observed fromthe nanowires are
highly photostable with an intensity level of 10 times larger than that observed fromsilver nanoparticles of comparable sizes. This intrinsic
optical signal enabledintravital imagingof nanowirescirculatingintheperipheral bloodof amouseandmappingof nanowiresaccumulated
intheliver andspleen, openingupfurther opportunitiestoinvestigateinvivocellular responsetonanomaterialsasafunctionof size, aspect
ratio, and surface chemistry.
The translation of nanomedicine to a clinical setting has been
slowed down due to limited fundamental understanding of
the interactions between nanomaterials and biological cells.1
Current study of cellular interactions with a nanomaterial is
challenged by the lack of a strong intrinsic signal from a
dimension-controllable nanostructure. Although routinely
used as labels for visualization of nanosized drug carriers,
such as liposomes, polymer particles, and copolymer mi-
celles, fluorescent agents often suffer from photobleaching.
The interpretation of fluorescence data could be further
complicated due to dissociation of probes from the objects
to be studied. For example, recent research revealed an
unexpected release of lipophilic dyes from copolymer
micelles2,3and poly(lactic-co-glycolic acid) nanoparticles4
to lipid-rich structures during cellular uptake or blood
circulation. Intrinsic near-infrared fluorescence5,6and spon-
taneous Raman scattering7have been used to track carbon
nanotubes in live cells and live animals, where the lengths
of nanotubes were limited to several hundred nanometers.
An intrinsic multiphoton luminescence8has been used to
visualize low-aspect-ratio gold nanorods and nanoshells in
live cells9and implanted tumors10while more than 95% of
the excitation energy is actually converted into heat, causing
effective phototoxicity to cells.9,11Therefore, in order to
decipher cellular response to nanostructures, nanosystems
with precise controls of size, shape, and surface chemistry
and an intense and intrinsic optical signal with low damage
potential are desired. Silicon nanowires (SiNWs) have
unparalleled dimension-control properties with diameters
controlled from ∼3 up to 100 nm,12,13and lengths from a
few hundred nanometers to tens of micrometers.14In this
paper, we report strong and stable third-order nonlinear
optical (NLO) signals, including four-wave mixing (FWM)
and third-harmonic generation (THG), from silicon nanowires
(SiNWs) of diameters as small as 5 nm. We further employ
such signals to monitor SiNWs circulating in the peripheral
blood of a live mouse and to map the organ distribution of
systemically administrated SiNWs.
SiNWs were synthesized by chemical vapor deposition of
silane using 40, 20, and 5 nm Au colloids (Ted Pella) as
catalysts.12For in vitro imaging, nanowires were transferred
onto coverslips through gentle friction between the as-grown
nanowire substrate and the coverslip, which also aligned
nanowires in a relatively parallel fashion. FWM and THG
images of SiNWs were acquired on a multimodal NLO
microscope with configurations as follows. A femtosecond
laser (Mai tai, Spectra-Physics, Fremont, CA) generated 130
fs pulse at a repetition rate of 80 MHz. Eighty percent of
the Mai Tai output at 790 nm was used to pump an optical
parametric oscillator (OPO) (Spectra-Physics, Fremont, CA)
generating a signal beam at 1290 nm and an idler beam at
2036 nm. The frequency of the idler beam was doubled to
1018 nm by a PPLN crystal. The 1018 nm beam was used
as the Stokes beam. The other 20% of the Mai Tai beam
was used as the pump beam. These two beams were
* To whom correspondence should be addressed. E-mail: yang@
†Department of Physics.
‡Department of Chemistry.
§Weldon School of Biomedical Engineering.
|These authors contributed equally to this work.
Vol. 9, No. 6
10.1021/nl901143p CCC: $40.75
Published on Web 05/06/2009
2009 American Chemical Society
collinearly combined with the Stokes beam passing through
a delay line. The combined beams were sent into a FV1000
laser-scanning microscope (Olympus America Inc., PA) and
focused into a sample using a 60× water objective lens with
a numerical aperture of 1.2. The backward NLO signal was
separated from the excitation laser by a dichroic mirror and
detected by an external photomultiplier tube after passing
through a bandpass filter (430/40 nm for THG, 650/40 nm
for FWM). Emission spectra were recorded with the internal
spectral detector of the microscope.
SiNWs of 40 nm diameters synthesized exhibit strong
emissions in both FWM and THG images (Figure 1a,b,
insets). The FWM and THG emission spectra recorded from
individual nanowires in the 400-680 nm region display a
peak at 645 nm (Figure 1a) and at 428 nm (Figure 1b),
respectively. These peak positions are in agreement with the
emission wavelengths of FWM, 645 nm, generated by
collinearly combined pump field (790 nm) and Stokes field
(1018 nm), and THG, 430 nm, produced by 1290 nm
excitation, confirming that the contrast in the images (Figure
1a,b, insets) arises from FWM and THG, respectively.
It is evident that the FWM signal is not resonant with the
Raman shift of approximately 520 cm-1observed previously
in SiNWs.15-17This vibrationally nonresonant yet strong
emission suggests the possibility of further enhancing the
FWM signal level by tuning the pump and/or Stokes laser
wavelengths. The intensive FWM and THG emission from
SiNWs could be attributed to the large third-order suscep-
tibility of crystal silicon, which is approximately 1-2 orders
of magnitude higher than that of other materials such as
crystal CdS, TiO2, and Au.18Notably, second harmonic
generation (SHG) signals in nanowires have been reported.19
Strong SHG response from potassium niobate (KNbO3)
nanowires partially resulted from its large nonlinear optical
coefficients enabled a nanometric light source when a single
KNbO3nanowire was optically trapped.20In our case, the
intensive intrinsic THG signals enabled visualization of
individual SiNWs with diameter as small as 5 nm (Support-
ing Information, Figure S1) and were employed for real-
time imaging in vivo. Additionally, the lateral and depth full-
width-at-half-maximum (fwhm) of the FWM signal measured
on a single nanowire were measured to be 0.30 and 1.43
µm (Supporting Information, Figure S2), respectively. Such
three-dimensional (3D) spatial resolution allowed us to
monitor individual SiNWs grown on a quartz substrate
(Supporting Information, Figure S3) or dispersed in a 3D
collagen scaffold (Supporting Information, Figure S4).
THG images of aligned SiNWs were recorded while
rotating the polarization of the excitation laser to 0, 30, 60,
and 90° with respect to the nanowire axis (Figure 2a). The
results show that the THG intensity was maximized when
the polarization of excitation was parallel with the nanowire
axis and almost depleted when the polarization of excitation
was perpendicular to the nanowire axis.19
As shown in Figure 2b, the THG intensity measured on a
representative nanowire exhibits a periodic dependence on
the angle θ. This periodic polarization dependence could be
modeled by treating the nanowire as an infinite dielectric
cylinder similarly as that in the previous photoluminescence
study of nanowires.21According to this model, the perpen-
dicularly polarized electric field inside the cylinder Einis
attenuated to 75% of the excited field Eebased on Ein)
Figure 1. Imaging NWs with NLO signals. (a) FWM image and
spectrum of SiNWs. The pump and Stokes laser power at the sample
were 0.8 and 1.2 mW, respectively. (b) THG image and spectrum
of SiNWs. The 1290 nm laser power at the sample was 8.6 mW.
All images and spectra were recorded in 0.5 s. Scale bars, 2 µm.
Figure 2. Polarization dependence of THG intensity from SiNWs.
(a) THG images of aligned SiNWs under the excitation polarization
0° (upper left), 30° (upper right), 60° (lower left), and 90° (lower
right) with respect to the nanowire axis. Scale bar, 2 µm. (b)
Measured THG intensity (solid squares) as a function of excitation
polarization angle relative to the nanowire axis. Red curve, a least-
squares fitting by cos6θ.
Nano Lett., Vol. 9, No. 6, 20092441
(2ε0)/(ε0+ ε)Ee, where ε and ε0are the dielectric constants
of silicon and vacuum.22As a result, the signal intensity
arising from the perpendicular polarization is much smaller
than that from parallel polarization. Distinguished from the
previous cos2θ dependence found in one-photon photolu-
minescence and Raman scattering of nanowires,21,23the
corresponding THG intensity is expected to show a cos6θ
dependence on the polarization of excitation, as the THG
field is proportional to the cube of the excitation field. This
cos6θ relationship is confirmed by the least-squares fitting
(red curve) shown in Figure 2b.
To evaluate its potential as a valid NLO imaging agent,
we compared the THG signal intensity and photostability of
single SiNWs with those of silver nanoparticles (NPs), one
of the strongest THG emitters studied previously.24,25We
continuously scanned 40-nm diameter SiNWs and 60-nm
diameter silver NPs for 70 s with 8.6 mW of the 1290 nm
beam at the sample. A strong THG intensity is expected from
silver NPs because the THG wavelength generated (430 nm)
is near the surface plasmon resonance wavelength of these
NPs. THG images acquired at the scanning time of 0, 22,
44, and 66 s show that the THG intensity of the SiNWs
remain consistent over the time (Figure 3a). In comparison,
THG signals from the silver NPs quenched quickly and few
signals from silver NPs were observed after 60 s (Figure
3b). The rapid decrease of the THG intensity is possibly due
to melting of the NPs by the ultrafast pulses. For quantitative
analysis of the intensity levels, the THG intensities of seven
SiNWs and seven silver NPs versus the scanning time are
plotted in Figure 3c,d, respectively. Under the same condition
of excitation, the THG intensity at the beginning of scanning
ranged from 600 to 2300 au for the SiNWs and from 200 to
450 au for the silver NPs. The intensity difference between
Figure 3. THG intensity and photostability of SiNWs and silver
NPs. (a) THG images of silicon NWs recorded at different scanning
time. (b) THG images of silver NPs recorded at different scanning
time. The scanning time is indicated in each image. Scale bars, 2
µm. (c) THG intensity of seven representative SiNWs as a function
of the scanning time. (d) THG intensity of seven representative
silver NPs as a function of the scanning time. The THG signals
were produced by the 1290 nm beam with 8.6 mW at the samples.
Figure 4. In vivo FWM images of SiNWs. (a) FWM image (red) of the peripheral blood of a living mouse taken at 20 min post injection
of PEGylated SiNW PBS solution. Yellow dashed lines mark the blood vessel. The white solid line indicates the scan line for the intensity
profile showed in b. (b) FWM intensity profile from the linescan along the flowing SiNW. (c) FWM image of the peripheral blood of a
living mouse taken post injection of PBS. (d-f) FWM images of SiNWs (red) deposited in liver (d), spleen (e), and kidney (f) explanted
at 1 h post injection. All FWM images are superimposed with transmission images (cyan) taken simultaneously. The SiNWs were highlighted
by yellow circles. Scale bars, 5 µm.
Nano Lett., Vol. 9, No. 6, 2009
individual nanowires is caused by the orientation variation.
For silver NPs, the intensity distribution can be due to various
aggregations. This comparison shows that the SiNWs
produced approximately ten times stronger THG than the
silver NPs. Results above demonstrate that SiNWs can be
rendered as an extremely intensive and stable NLO imaging
The potential of SiNWs in intravital imaging were
demonstrated for the first time by real time imaging of
polyethylene glycol (PEG)-modified SiNWs circulating in
the blood vessels inside a mouse earlobe. PEGylation has
been found to be able to prolong the blood circulation time
for other nanosystems, such as liposomes,26Au nanorods,27
and carbon nanotubes,28thus modification of SiNWs with
PEG are expected to promote circulations of SiNWs in the
blood to facilitate the imaging. We introduced 100 µL of
phosphate buffered saline (PBS) containing 0.1-1 pM
PEGylated SiNWs29of ∼5 µm in length to an anesthetized
BALB/c mouse through tail vein injection. The laser beam
was focused on the ear lobe using a 40× water-immersion
objective. The laser power at the sample was 23 mW for
the pump beam and 2 mW for the Stokes beam. The
backward FWM signal was detected by an external photo-
multiplier tube with bandpass filters of 645/40 nm. The blood
vessel and surrounding tissues were visualized by transmis-
sion illumination and the circulating SiNWs were monitored
by epi-detected FWM simultaneously with scanning rate of
2 µs/pixel and 256 × 256 pixels/frame (Supporting Informa-
tion, video 1). One frame is shown in Figure 4a. The FWM
signal presents an elongated shape with approximately 5 µm
in the elongated direction, which is consistent with the length
of SiNWs synthesized. Additionally, the FWM intensity
profile across the flowing SiNW (Figure 4b) shows a peak
intensity of 1500 au, which is 5 times larger than the
background (∼300 au) from the blood. Such FWM signal
was not detected in the control mouse injected with 100 µL
pure PBS (Figure 4c). After 30 min post injection, we could
no longer detect any FWM signals from the SiNWs in the
bloodstream. Compared to blood circulation time ranging
from 1.5 to 15 h observed for single carbon nanotubes with
various PEGylations,7the shorter circulation time of SiNW
could be due to the shorter PEG chain used and/or dimension
difference between SiNWs and carbon nanotubes. Further
systematic investigation will be carried out to address these
Using the FWM signals, we further studied the distribution
of SiNWs in the organs explanted at 1 h post injection. To
prepare explanted organ tissues, the mouse was euthanatized
at 1 h after intravenous injection of SiNWs. Organs including
liver, spleen and kidney were explanted, fixed in 4% formalin
solution to preserve the tissue architecture, and cut into small
pieces by blade for imaging. The laser beams were focused
onto the sample using a 60× water-immersion objective with
a laser power of 10 mW for pump beam and 3 mW for
Stokes beam at the sample. FMW images superimposed with
transmission images taken simultaneously show that the
SiNWs appearing wire shapes were found in both liver
(Figure 4d) and spleen (Figure 4e) tissues. The depth-
resolved distributions can be found in the Supporting
Information, video 2. No SiNWs were observed in the kidney
(Figure 4f). These results suggest that most of the injected
SiNWs were captured by the reticuloendothelial system but
not filtered through kidney. It is conceivable that the SiNWs
were captured by the macrophages while circulating through
the liver and spleen.
In summary, we have demonstrated that SiNWs exhibit
intensive FWM and THG emissions with a cos6θ polariza-
tion dependence. These properties open up exciting op-
portunities for using SiNWs as a novel in vivo imaging agent
offering intrinsic 3D spatial resolution, high photostability,
and orientation information. With the advantages of highly
controllable dimensions, versatile surface chemistry, and an
intensive intrinsic NLO signals, SiNWs provide an exciting
nanobio system for investigating the cellular interaction with
Acknowledgment. The work was supported by start-up
funds from Purdue University, NSF Grant CBET 0828832,
and American Heart Association predoctoral fellowship for
Supporting Information Available: Depth-resolved FWM
images of SiNWs spread on a coverslip (Figure S1).
Reconstructed 3D THG image of as-grown 5 nm SiNWs on
a quartz substrate (Figure S2). SiNWs embedded in a
collagen gel served as a tissue scaffold (Figure S3). THG
images of SiNWs with diameters of 40, 20, and 5 nm (Figure
S4). In vivo FWM imaging of SiNWs circulating in a blood
vessel recorded at 15 min after injection (Supporting Video
1). Depth-resolved FWM imaging of SiNWs deposited in
spleen explanted at 1 h postinjection (Supporting Video 2).
This material is available free of charge via the Internet at
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(29) To prepare PEGylated nanowires, SiNW growth substrates were first
modified with 1% v/v 3-(trimethoxysilyl)propyl aldehyde in ethanol
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Nano Lett., Vol. 9, No. 6, 2009