ISSN:1369 7021 © Elsevier Ltd 2011JUNE 2011 | VOLUME 14 | NUMBER 6264
Label-free imaging through nonlinear optical signals
Nonlinear optics describes the behavior of light in a medium where
the dielectric polarization P responds nonlinearly to the electric
field E of the light. Examples of these processes include multiphoton
luminescence, second harmonic generation (SHG), third harmonic
generation (THG), four-wave mixing (FWM), coherent anti-Stokes
Raman scattering (CARS), pump-probes, and the photothermal
effect (Fig. 1). Many nanostructures exhibit strong intrinsic nonlinear
optical (NLO) signals under tight focusing conditions. Combining
NLO signals with scanning microscopy has generated an array of
imaging modalities for material and biological studies1,2. Unlike
linear optical microscopy, NLO microscopy offers inherent 3D spatial
resolution, relatively large optical penetration into tissues with near
infrared (NIR) excitation, and reduced photodamage due to reduced
optical interaction with endogenous molecules. Many NLO contrasts
have been discovered across the broad spectrum of nanomaterials, as
summarized in Table 1.
Strong intrinsic nonlinear optical (NLO) signals not only make nanostructures promising agents for bio-imaging, but also advance NLO microscopy for the study of interactions between nanomaterials and live cells. Single beam modalities such as multiphoton luminescence, second harmonic generation, and third harmonic generation provide a simple way to probe many types of nanostructures. As for more advanced modalities, photothermal heterodyne imaging provides improved detection sensitivity for smaller objects, and transient absorption microscopy provides structural information to distinguish metal from semiconducting carbon nanotubes, and eumelanin from pheomelanin. The four-wave mixing signal achieves chemical selectivity in the presence of either vibrational or electronic resonance, as used in coherent Raman scattering imaging of molecules and in electronically resonance enhanced four-wave mixing imaging of nanostructures.
Ling Tonga and Ji-Xin Chenga,b* aDepartment of Chemistry, Purdue University, West Lafayette, IN 47907, USAbWeldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA
*E-mail: [email protected]
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In this article, we review recent advances in the exploration
of intrinsic NLO signals from metallic and semiconducting
nanostructures. We also review how NLO signals can be applied to
provide an imaging contrast for the study of cellular interactions with
nanomaterials.
Multiphoton luminescence from gold nanostructuresFirst reported by Mooradian in 196962, photoluminescence from
noble metals is generated in a three-step process: (i) excitation of
electrons from the d- to the sp- band to generate electron-hole pairs,
(ii) scattering of electrons and holes on the picosecond timescale with
partial energy transfer to the phonon lattice, and (iii) electron-hole
recombination resulting in photoemission. Photoluminescence can
be generated by single or multi-photon excitation, and the efficiency
can be enhanced by several orders via resonant coupling with
localized plasmons from nanostructures63,64. Herein we focus on the
multiphoton luminescence from gold nanostructures.
Two-photon luminescence Two-photon luminescence (TPL) from nanostructured gold was first
demonstrated by Boyd et al. in 198664, but the efficiency from the gold
film was low. With the longitudinal plasmon resonance peaks shifted to
NIR wavelengths, gold nanorods (GNRs)6,7, gold nanoshells8 and gold
nanocages12 are particularly well suited for use as TPL-based imaging
agents. The mechanism and characteristics of TPL from GNRs have
been investigated by both far-field6,65 and near-field5,7 microscopy. A
broad luminescence emission from 400 – 650 nm has been observed
in both cases6,7, corresponding to electron-hole recombination near
the X and L symmetry points of the Brillouin zone. In the far field
study, Wang et al.6 investigated the relationship between TPL and
the longitudinal plasmon resonance mode of GNRs. The excitation
spectrum overlaps with the longitudinal plasmon band, indicating that
plasmon-enhanced two-photon absorption contributes to the TPL from
GNRs. Due to the rod shape, TPL from a single GNR is sensitive to the
polarization of the incident beam, as shown in Figs. 2a,b. The individual
Fig. 1 Nonlinear optical (NLO) modalities employed for the visualization of nanomaterials. Solid lines represent electronic and vibrational states of molecules. Dashed lines are virtual states. The straight and wavy arrows are excitation and output signal beams, respectively. The gray arrows represent relaxation in electronically excited states. ω1 and ω2 are excitation beams. ωp and ωs are pump and Stokes beams for CARS. Ω is the frequency of vibrational transition between vibrational ground state and vibrationally excited state. ωp and ωpr are the pump and probe beams for pump-probe and photothermal modalities. ΔT is the temperature change due to excitation. n1 and n2 are the refractive indices of the material before and after excitation, respectively. TPEF: Two-photon excited fluorescence. TPL: Two-photon luminescence. SHG: Second harmonic generation. THG: Third harmonic generation. 3PL: Three-photon luminescence. CARS: coherent anti-Stokes Raman scattering. FWM: Four-wave mixing.
NLO modalities Nanomaterials
Multiphoton luminescence from gold nanostructures Gold nanoparticles3,4, gold nanorods5-7, gold nanoshells8, gold nanowires9,10, and Au-Ag nanocages11,12.
SHG ZnO nanowires13, GaN nanowires14, KNbO3 nanowires15, metallic nanoparticles16-18, nanocrystals19-22, and quantum dots23.
THG Gold nanoparticles24, silver nanoparticles25, 26, gold nanorods27, Si nanowires28, ZnO nanowires13, ZnO thin films, CdSe quantum dots, and Fe3O4 nanoparticles29.
FWM Semiconducting and metallic nanostructures30 (e.g., gold nanowires31, gold nanoparticles32, gold nanoparticle antennas33, gold nanorods34, Si nanowires28, TiO2, ZnO, CeO2, Fe2O3 nanoparticles35-37, quantum dots38 ), SWNT39, and graphene flakes40.
CARS Polymeric nanoparticles41,42.
Transient absorption Semiconducting and metallic nanostructures41,42 (e.g., gold and silver nanoparticles41,43-45, nanoparticle dimers46, CdTe nanowire47, and silver nanowire48), and SWNT49.
Photothermal imaging Metal nanoparticles (e.g., gold nanoparticles50-58, gold nanorods59), quantum dots60, and SWNT61.
Table 1 Nanomaterials investigated by NLO microscopy. (SWNT: single-walled carbon nanotube).
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nanorods exhibit the same TPL intensity level with horizontally or
vertically polarized excitations (spots 1 & 2), whereas nanorod clusters
emit a brighter signal (spot 3). Wang et al. found that the TPL intensity
from single nanorods follows a cos4 function of the polarization angle
of the incident beam (Figs. 2c), in accord with a coherent two-photon
absorption process and the dipolar nature of the nanorods’ plasmon
resonance. Interestingly, the TPL emission is found to be depolarized
(Fig. 2d), similar to SHG but in contrast to the conventional plasmon-
resonant scattering process66. In the near-field microscopy study by
Okamoto and co-workers5,7, a cos2 function has been demonstrated on
the polarization of incident excitation, which is explained by describing
the two-photon excitation as sequential one-photon processes. With
high spatial resolution provided by scanning near-field microscopy,
an oscillating pattern has been observed in GNRs5,7. More recently,
Nan et al. observed a spatially modulated TPL from nanowires
composed of a silicon core with a gold shell by far field microscopy67.
These patterns are interpreted as the periodic amplitude modulations
of the wavefunctions for the standing-wave plasmon modes.
The TPL signal from a single GNR was found to be nearly 60 times
brighter than the two-photon fluorescence signal from a rhodamine
molecule6. Such strong TPL signals have been widely used in biomedical
studies68 to map the bioconjugated GNRs in cellular uptake and
intracellular trafficking69,70 (Fig. 2e), to monitor the photothermal
damage process69, to determine biodistributions71,72 (Fig. 2f), and to
track systemic clearance in the blood of live animals72. For example,
real-time TPL imaging enabled Tong et al. to monitor membrane
blebbing during GNR (45 nm in length, aspect ratio of 3.7) induced
photothermal damage of targeted tumor cells, and to decode the
cell death mechanism at the sub-cellular level69. Through intravital
TPL imaging of GNRs flowing in the blood vessels of live mice,
Tong et al. quantitatively analyzed the clearance of PEGylated GNRs
using a biphasic model, and studied the impact of surface coating on
pharmacokinetics72 (Figs. 2g,h). Unlike traditional methods of studying
the clearance of nanoparticles73, in which animals need to be sacrificed
or blood need to be extracted at each post-injection time point,
intravital TPL microscopy is able to track nanoparticles in the same
animal without cumbersome sample preparation. As another important
application, the plasmon-medicated TPL from GNRs has been used to
realize five-dimensional optical recording with enhanced wavelength
and angular sensitivity, showing high potential for optical patterning,
encryption, and data storage74.
Three-photon luminescenceCompared to plasmon resonance enhanced TPL, three-photon
luminescence (3PL) was found at an off-resonance excitation.
Farrer et al.4 and Eichelbaum et al.3 demonstrated 3PL from gold
Fig. 2 Two-photon luminescence (TPL) from gold nanorods (GNRs). (a,b) Pseudocolor images with horizontal (θ = 0°) and vertical (θ = 90°) excitation polarizations, respectively. Single nanorods could be identified by their similar range of intensities (e.g., spots 1, 2); brighter spots were considered to be clusters of nanorods (e.g., spot 3). (c) Polarization dependence of the TPL intensity (solid dots) for a single nanorod (spot 2). The excitation polarization was rotated clockwise from -90° to +90° in 10° increments by controlling a halfwave plate. The TPL signal fitted a cos4 function with an offset of 6.8° (red curve). (d) The TPL emission (solid dots) from the same nanorod (spot 2) measured by rotating a polarizer before the detector. The reflected excitation beam was linearly polarized at the detector site (solid curve), but the TPL emission was essentially depolarized. Reprinted from6. © National Academy of Sciences. (e-h) TPL imaging of bioconjugated GNRs in vitro and in vivo. (e) TPL imaging of folate conjugated GNRs (red dots) internalized by KB cells © Wiley-VCH Verlag GmbH & Co. KGaA. Reproduces with permission from69. (f) TPL imaging of GNRs (in green circle) accumulated in liver after systemic clearance. Hepatocytes were visualized by autofluorescence. (g) Intravital TPL imaging of PEGylated GNRs circulating in a blood vessel (outlined by the dash yellow line) in the earlobe of a living mouse. The image was compiled by stacking the 60 frames collected continuously at a rate of 0.4 s/frame in a video taken at 10 min after injection of nanorod solution. (h) Quantitative analysis of the systemic clearance of GNRs based on the TPL contrast. The blood concentration of GNRs coated with branched PEG7K was represented by INR/Itissue. Each data point represents an average from 3 mice (n = 3). The data was fitted with a one-compartment model (red) and two-compartment model (green). Length bar = 10 μm. (h-f) Reprinted with permission from72. ©2009 American Chemical Society.
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nanoparticles though excitation with a femtosecond (fs) pulsed
laser around 790 nm. It has been suggested that the enhancement
was related to the high density of states around 267 nm. Recently,
Tong et al. reported bright 3PL from Au-Ag alloyed nanostructures by
exciting at 1290 nm11. The photoluminescence demonstrated a broad
emission profile between the THG peak at 430 nm and the SHG peak
at 645 nm (Figs. 3a,b), and a cubic dependence of intensity on the
excitation power. The 3PL signal from Au-Ag alloyed nanostructures,
including hollow and porous nanocages and solid nanoparticles, was
nearly one order of magnitude brighter compared to pure gold or silver
nanoparticles. Because an excitation at 1290 nm is away from the
plasmon resonance, the three-photon absorption cross-section should
be negligible when compared to the parametric THG process for Au-Ag
nanostructures, whose third-order susceptibility (χ(3)) is larger than pure
silver nanoparticles measured by the Z-scan technique75. An alternative
mechanism has also been proposed to explain the enhanced 3PL, that
is, the generated THG photons after the 1290 nm excitation might be
re-absorbed by the same particle to produce the luminescence11.
Because the excitation at 1290 nm is away from the plasmon
resonance of Au-Ag alloyed nanostructures, the intense 3PL enables
bio-imaging with undetectable photothermal toxicity and lower tissue
autofluorescence11. Unlike plasmon resonant TPL processes in which
most of incident photons are absorbed by plasma and then converted
into heat, in the 3PL case with off-resonance excitation, absorbed
photons are more efficiently used for generating luminescence. As
shown in Figs. 3c-f, Au-Ag nanocages (~40 nm in edge length) inside
KB cells produced the same intensity level by TPL excited with a
760 nm laser at 1.9 mW and by 3PL excited with a 1290 nm laser
at 4.0 mW. After continuous scanning of the same cell for 90 s in
both cases, cell death (indicated by ethidium bromide staining) and
decreased TPL intensity from the Au-Ag nanocages were observed in
the cell irradiated by the 760 nm laser; whereas neither cell damage
nor reduction of 3PL intensity were detected in the cell scanned by the
1290 nm laser. For tissue imaging, the absence of autofluorescence
from excitation at 1290 nm makes 3PL easier than TPL to map
and determine the amount of Au-Ag nanocages distributed in the
tissues (Figs. 3g,h). The intrinsic and bright 3PL makes Au-Ag alloyed
nanostructures attractive imaging agents for long-term monitoring of
nanoparticles in biological environments.
Second harmonic generation (SHG)SHG is a special case of sum frequency generation, in which photons
(ω1) interact with the NLO media and are converted into new photons
with twice the energy (2ω1). As a second order NLO process, only
noncentrosymmetric structures are able to emit SHG signals. Since the
first demonstration in 1961, SHG has been used to probe interfaces,
Fig. 3 Three-photon luminescence (3PL) from Au-Ag nanocages. (a) Emission spectrum from a Au-Ag nanocage (49 % Ag – 51 % Au) excited by a 1290 nm laser. (b) 3PL image of Au-Ag nanocages (49 % Ag – 51 % Au) spin coated on a coverglass. (c-h) Comparison of 2PL and 3PL imaging of Au-Ag nanocages in (c-f) KB cells and (g,h) liver tissues. (c) 2PL image and (d) 3PL image of Au-Ag nanocages (red) in KB cells before laser scanning. (e) Image of the same cell in (c) after scanning with a 760 nm fs laser for 90 s. The laser power after the objective was 1.9 mW. After scanning, membrane blebbing (arrowed) and compromised membrane integrity indicated by ethidium bromide labeling (green) were observed. (f) 3PL image of the same cell in (d) after scanning with a 1290 nm fs laser for 90 s. The laser power after objective was 4.0 mW. No morphological change or plasma membrane damage was observed. (g) 2PL imaging of Au-Ag nanocages (indicated by white circles) in liver tissue. White arrow: Anomalously strong autofluorescence from tissue. (h) 3PL imaging of Au-Ag nanocages (indicated by white circles) in liver tissue at the same area as (g). For both 2PL and 3PL imaging of nanocages in liver, the laser power was 7 mW after the objective. Composition of the Au-Ag nanocages: 49 % Ag and 51 % Au. Length bar = 10 μm.© Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission from11.
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in the diagnosis of diseases76,77 and for label-free imaging of ordered
structures in cells, tissues, and organisms (such as microtubules,
collagen, and muscle myosin)78,79. Finazzi et al. discussed the selection
rules for SHG in isolated nanoparticles based on the angular momentum
and parity conservation laws80. Strong SHG signals have been found
in many types of single inorganic nanostructures, including ZnO
nanowires13, GaN nanowires14, KNbO3 nanowires15, noble metallic
nanoparticles16-18, nanocrystals (e.g., Fe(IO3)3, KTiOPO4, BiTiO3)19-22,
as well as core/shell CdTe/CdS quantum dots as small as 10 – 15 nm in
diameter23. The intense SHG signals were used for nanoscale imaging15
and to track nanoparticles in cells, tissues, and organisms21,22,81. For
example, Kuo et al. utilized SHG signals to monitor ZnO nanoparticles
in transdermal delivery81. Pantazis et al.22 and Hsieh et al.21
characterized SHG signals from BaTiO3 nanocrystals and developed
bioconjugated nanocrystals as probes for cell and in vivo imaging
without photobleaching or blinking. After an injection of BaTiO3
nanocrystals (~30 nm) into the zebrafish embryo, bright SHG signals
could be detected in either superficial tissue or deep within organs
in the zebrafish during development. Unlike fluorescent dyes, the
SHG nanoprobes exhibit a narrow emission profile, resulting in a high
signal-to-noise ratio (SNR) in the tissues with little autofluorescence
background22. Based on the intense SHG signals from BaTiO3
nanocrystals, a harmonic holographic microscope has been developed
for 3D imaging of nanocrystals in cells without scanning82.
Third harmonic generation (THG)THG is a third-order NLO process that requires three photons (ω1)
in order to generate one photon at the tripled frequency (3ω1).
Unlike SHG, THG is not restricted to noncentrosymmetric structures.
Under the microscopy conditions, the THG signal generated from a
homogenous medium vanishes due to the Gouy phase shift of the
excitation field across the focus83,84. As a result, THG microscopy is
sensitive to interfaces85 and has been used to image the interface of
a transparent specimen86. Because of its capabilities in 3D sectioning
and the relatively high damage threshold, the application of THG
microscopy has been extended to visualize biological samples in vitro
and in vivo without labeling87-90. The unique optical properties
of nanostructures render them ideal THG enhancing agents. For
example, the surface plasmon resonance from silver (e.g., 5 – 7 nm
nanoparticle array26, 30 nm nanoparticle25) and gold nanostructures
(40 nm nanoparticles24, gold nanorods with a length of 25 nm and
aspect ratio of 3.727) enhances the THG process. Semiconducting
nanomaterials also exhibit THG signals. Chang et al. demonstrated
backward THG signals from ZnO thin films, CdSe quantum dots, and
Fe3O4 nanoparticles29. Using a fs pulsed laser at 1290 nm, Jung et al.28
discovered a strong THG signal from Si nanowires as small as 5 nm
in diameter due to the large χ(3) from crystalline Si, which is 1 – 2
orders of magnitude higher than materials such as crystalline CsS, TiO2,
and gold83 (Fig. 4a). Compared with silver nanoparticles, one of the
Fig. 4 Third harmonic generation (THG) and four-wave mixing (FWM) signals from Si nanowires. (a) THG image and spectrum from Si nanowires, excited by a 1290 nm laser with laser power of 8.6 mW at sample. (b-c) THG images of Si nanowires acquired at different scanning time. (d) FWM image and spectrum from Si nanowires. The laser power of pump (at 790 nm) and Stokes (at 1018 nm) beams were 0.8 and 1.2 mW at the sample, respectively. (e-f) FWM imaging of Si nanowires in the biological environment. (e) FWM imaging of PEGylated Si nanowires (red) flowing in a blood vessel (outlined by dashed yellow line) in the earlobe of a living mouse. (e) FWM imaging of Si nanowires (red) in liver after systemic clearance in a mouse. The blood vessel and liver tissues were visualized by transmission images (cyan). Length bar = 5 μm. Reprinted with permission from28. © 2009 American Chemical Society.
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strongest THG emitters previously reported25,26,91, Si nanowires, exhibit
stronger and more photostable THG signals (Fig. 4b,c)28. The intense
THG signal has been used for label-free imaging of nanostructures in
cells and tissues25, 92.
Four-wave mixing (FWM) FWM is a third-order NLO process which is sensitive to χ(3). In FWM
microscopy, three incident laser fields with frequencies of ω1, ω2, ω3
interact with the material to generate a signal field at a frequency of
ω4. In principle, the FWM signal is sensitive to both the vibrational and
electronic properties of the material. In techniques using vibrationally
enhanced FWM, such as coherent anti-Stokes Raman scattering
(CARS)93,94, stimulated Raman scattering (SRS)95-97 and optical Kerr
effect microscopy98, the signal is enhanced when the wavenumber
difference between the incoming beams matches the molecular
vibration. CARS99,100 and SRS95,101-103 imaging have found widespread
application in biological studies. In electronically enhanced FWM,
one of the beams, or the combination of beams, is resonant with the
electronic transition of the material, and the FWM signal intensity is
significantly increased, especially in nanomaterials30.
Various nanomaterials have been demonstrated to act as FWM
imaging agents. Contributed by the localized surface plasmons,
FWM signals are enhanced in metallic nanostructures, such as gold
nanowires31, GNRs (~45 nm in length, aspect ratio of 3.6)28, gold
nanoparticles (10 nm)32, and gold nanoparticle antennas33, and
have therefore been applied to biological imaging28,32,34. A panel of
semiconducting nanomaterials, including metal oxide nanoparticles
in biological samples (e.g., 20 – 70 nm TiO2, ZnO, CeO2, Fe2O3
nanoparticles)35-37, quantum dots, and quantum wires38, and elemental
nanostructures (e.g., Si)28, also exhibit strong FWM signals. Jung et al.28
detected a strong FWM signal from Si nanowires (Fig. 4d) and applied
the intrinsic signal as imaging contrast to visualize Si nanowires in a
biological environment. Using FWM imaging, Jung et al. monitored
blood circulation of PEGylated Si nanowires in live animals and
mapped the distribution of Si nanowires in organs after systemic
clearance (Figs. 4e,f). Besides the examples above, FWM has been
detected in individual single-walled carbon nanotubes (SWNTs),
especially metallic SWNTs39 and graphene flakes40. FWM microscopy
not only allows characterization of the nonlinear susceptibility and
electron dynamics of individual nanostructures, but also provides a
powerful imaging modality utilizing an intense signal that is not prone
to photobleaching.
Coherent anti-Stokes Raman scattering (CARS)CARS is a FWM process in which a pump beam (ωp) and a Stokes
beam (ωs) interact with a sample to generate a signal at the anti-
Stokes frequency ωas = 2ωp – ωs93,94. The CARS signal is significantly
enhanced when ωp – ωs is close to the vibrational frequency (Ω) of
a chemical bond, making CARS microscopy a chemically selective
imaging tool that has been widely used in biological studies69,70.
Because the coherent addition of the radiation fields builds up a
large signal from the focal volume, CARS is particularly sensitive to
lipid particles or polymer-based nanocarriers due to their abundant
C-H bonds. The advantages of CARS microscopy have been utilized
in real-time visualization of the intracellular behavior of drug
nanocarriers and understanding the mechanism of the intracellular
drug release process. For example, to overcome the photobleaching
problem in fluorescence microcopy, Tong et al. developed a polymeric
nanoparticle-based CARS probe (200 nm in diameter) to study the
folate receptor mediated endocytosis and track intracellular trafficking
of the nanoparticles in the long-term104. Using label-free CARS
imaging, Xu et al. reexamined the cellular uptake pathway of poly
(lactic-co-glycolic acid) (PLGA) nanoparticles and discovered a new
mechanism for PLGA nanoparticle-based drug delivery system105.
Through direct visualization of the PLGA polymer, Xu et al. observed
separation of the loaded dye molecules from the PLGA nanoparticles
(300 nm in diameter) during interactions with cells, suggesting the
previous data based on the fluorescent imaging of the dye-loaded
PLGA nanoparticles overestimated the internalization rate of PLGA
nanoparticles. Coupling CARS with other NLO modalities such as TPEF
can be used to monitor drug release from the carriers in real time.
Besides CARS, recently developed SRS microscopy95-97 is free of the
non-resonant background and is more sensitive to low concentration
molecules. Applications of SRS microscopy for the characterization of
nanomaterials are anticipated.
Transient absorption Absorption is an optical property that has been extensively used for
label-free imaging. Transient absorption, a time-resolved technique, has
shown great potential in selective chemical mapping of nanomaterials
and compounds. In transient absorption, an intensity-modulated pulsed
laser pumps a molecule or nanostructure to an electronically excited
state. The second pulsed laser probes the difference in absorption
between the ground state (pump field off) and the excited state (pump
field on). The modulation of the probe beam intensity is measured
by a lock-in amplifier. Varying the delay between pump and probe
fields provides information about the metastable states or transient
species106. Several mechanisms contribute to the pump-probe transient
absorption signal: multiphoton absorption, excited state absorption,
stimulated emission, and optical bleaching.
Transient absorption microscopy offers an excellent contrast for
mapping non-fluorescent or weakly fluorescent molecules in biological
systems. As shown by Warren and co-workers, eumelanin and
pheomelanin (two types of melanins) in human melanoma lesion, skin,
and hair can be selectively visualized among other fluorophores107-110.
Eumelanin and pheomelanin could be distinguished by their different
phases in transient absorption signal. The vasculature, particularly
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the oxygen level in blood, could be detected by in vivo transient
absorption imaging with different signal levels for oxy- and deoxy-
hemoglobin111,112. Stimulated emission detection of nonfluorescent
chromoproteins was shown by Xie and co-workers113.
Transient absorption spectroscopy and microscopy have also been
applied to study the ultrafast electron dynamics in nanomaterials42. The
first application of this for a single nanoparticle was performed by the
Orrit group using a birefringent crystal in an interferometer114. Others
investigated the sub-picosecond dynamics in single semiconducting
and metal nanostructures, such as gold and silver nanoparticles41,43-45,
nanoparticle dimers46, CdTe nanowire47, and silver nanowire48.
These measurements provide information about electron-photon
coupling, energy dissipation, charge carrier trapping, and dephasing of
vibrational resonances inside the nanostructures. Transient absorption
measurement have also been applied to SWNTs. The electronic
structure and ultrafast relaxation process of bulk SWNTs in solutions or
films has been investigated by transient absorption spectroscopy115,116.
Most recently, Jung et al. demonstrated high-speed mapping of the
metallic properties of individual SWNTs by transient absorption
microscopy49. With the pump and probe wavelengths tuned to 707
and 885 nm, semiconducting SWNTs (S-SWNTs) and metallic SWNTs
(M-SWNTs), which have different energy structures, exhibited positive
in-phase signals and negative anti-phase signals, respectively. Using
the aligned single SWNTs on quartz substrates and by comparison with
atomic force microcopy images, Jung et al. demonstrated the ability to
detect individual SWNTs with submicron resolution (Figs. 5a-c). The
strong signals were not attributed to the SRS of the G-band (Figs. 5d,e).
By investigating the dependence of transient absorption signals from
SWNTs on the pump/probe energies, it is anticipated that a stimulated
emission process is involved in the in-phase signal from S-SWNTs, and
that a photon-induced broadening of the transition width contributes
to the anti-phase signals from M-SWNTs. This study not only provides
a promising tool for mapping the metallicity of SWNTs in nanoelectric
devices, but also overcomes the challenge of visualizing both S-SWNTs
and M-SWNTs in biological studies.
Photothermal imagingUpon optical excitation of a non-fluorescent molecule or nanostructure,
energy rapidly distributes among many vibrational modes, and
eventually transfers to the surrounding medium as local heating.
This photothermal effect has been widely used for both therapy and
imaging. Photothermal imaging is a two-color NLO technique, in
which the pump (heating) beam is absorbed by the sample, resulting
in a thermal expansion and a localized change of refractive index.
The propagation of the probe beam at a different wavelength is then
modulated by the photothermal effect. The modulation is detected
with a lock-in amplifier. The mechanism of the photothermal signal
offers the following advantages: firstly, the signal arises from the
absorbing centers in a label-free manner. Secondly, the signal could
be enhanced by choosing a probe wavelength out of the absorption
Fig. 5 Transient absorption imaging of aligned individual SWNTs. (a) Schematic showing aligned SWNTs grown on a quartz substrate and the excitation and collection of light. (b) Atomic force microcopy (AFM) image of aligned SWNTs indicated by (1), (2), and (3). Inset: height profiles for each SWNTs. (c) Transient absorption image of SWNTs at the same location as in the AFM image with the pump and probe beams at 707 nm and 885 nm, respectively. The SWNT at the position (1) with anti-phase signal was identified as a M-SWNT, while the SWNTs at the position (2) and (3) with in-phase signals were identified as S-SWNTs. (d-e) Independence of signal on the Raman shift. Transient absorption image of a single S-SWNT at the Raman shift of 1587 cm-1 (d) and 1416 cm-1 (e). Length bar = 2 μm. Reprinted from49. © 2010 by the American Physical Society.
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band, and by increasing the probe power. Thirdly, photothermal
detection is especially sensitive to small objects for which absorption
dominates the scattering58, and is able to measure the absorption
spectral profile and the absorption cross-section of individual
nanoparticles58. The method has been used to detect the absorption
of single nonfluorescent azo dye molecules117, and to directly visualize
biological components, such as mitochondria in live cells118 and red
blood cells in live animals119.
Photothermal microscopy has also been widely applied to study
the nanostructures50,120. The first photothermal imaging of nanosized
absorbers, in this case 2.5 nm diameter gold nanoparticles, was
demonstrated by Boyer et al. using a photothermal interference
contrast method57. In their work, the signal was generated by a phase
shift between two orthogonally polarized and spatially separated
beams of an interferometer, with only one beam propagating through
the heated region. Later, the sensitivity was improved by two orders of
magnitude through use of the photothermal heterodyne imaging (PHI)
method121. In PHI, the probe beam generates a modulated scattered
field when it interacts with the time-modulated variations of the
refractive index around the sample caused by the time-modulated
pump beam. The scattered field is then detected by a lock-in
amplifier with the probe field as a local oscillator. The theoretical
framework of PHI is discussed and compared with experimental data53.
Photothermal spectroscopy and microscopy has been applied to metal
nanoparticles55,58,59, quantum dots60, and carbon nanotubes61. For
example, photothermal spectroscopy has been used to estimate the
concentration of nanosized particles in solvents122 and to measure the
absorption of individual SWNTs61. By the far-field polarization sensitive
photothermal imaging, Link and co-workers probed the anisotropic
absorption properties of small GNRs and measured the nanorod
orientation based on the transverse plasmon mode which is difficult to
detect with other optical imaging tools59. Recently, the detection limits
of photothermal microscopy have been discussed and the SNR has
been optimized as a function of probe power, as well as the optical and
thermal properties of the embedding medium of the absorbers, and the
isolation of the absorbers from the glass substrate50. Examples of PHI
imaging of 20 nm and 5 nm gold nanoparticles are shown in Figs. 6a-d.
With the development of photothermal correlation spectroscopy, the
diffusion of single gold nanoparticles, gold-protein complex, and gold
labeled bacteriophage has been investigated123-125.
Because the photothermal signal from nanostructures is not prone
to photobleaching or blinking, it has also been used for sensing and
imaging in biological applications. Blab et al.52 demonstrated the
potential of PHI as a reliable quantification of DNA hybridization in
gold nanoparticle-labeled DNA microarrays as it has a high sensitivity
and a dynamic range. Kulzer et al. applied photothermal detection
of individual gold nanoparticles in high-throughput screening51.
Cognet et al.56 visualized membrane proteins labeled with 10 nm gold
nanoparticles in cells by photothermal imaging, providing an efficient
and reproducible alternative to traditional electron microscopy.
Fig. 6 Photothermal imaging of gold nanoparticles. (a-d) Characterization of photothermal signals from gold nanoparticles. Reproduced from50 by permission of The Royal Society of Chemistry. (a) Photothermal microscopy image of 20 nm and 5 nm gold nanoparticles (marked with circles) immobilized on a glass slide in glycerol. The signal is color-coded on a logarithmic scale. (b) Histograms of the photothermal signal-to-noise ratios (SNRs) of gold nanoparticles. The population of 5 nm and 20 nm nanoparticles have SNR =12 ± 4 and SNR = 421 ± 92, respectively. (c) Photothermal image of a single 20 nm gold nanoparticle. (d) Photothermal image of a single 20 nm gold nanoparticle taken along the vertical z-axis, perpendicular to the glass surface. Gaussian fits of the shape of the signal along the z-, y-, and x-axes gives FWHM values of 730 nm, 250 nm, and 220 nm, respectively. Vertical dashed lines indicate the position of the glass-glycerol interface (glass on the right hand). (e-g) Photothermal imaging and single particle tracking of gold nanoparticles in a live neuron. Reprinted from54, Copyright 2006, with permission from Elsevier. (e) White light image and (f) PHI image of a live neuron labeled with gold nanoparticles. PHI image exhibits signals from one stationary (spot) and two moving (strips) membrane receptors labeled with gold nanoparticles (arrows). (g) Trajectory of an individual 5 nm gold nanoparticle (5 min, 9158 data points) acquired at video rate on a live neuron.
(a) (b) (e) (f)
(c) (d)(g)
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JUNE 2011 | VOLUME 14 | NUMBER 6272
Applications of PHI have been extended to track single nanoparticles
in live cells by Lasne et al.54. The authors overcame the challenge of
slow imaging speed in normal photothermal microscopy by designing
a single particle photothermal tracking scheme. Using this system,
Lasne et al. were able to track membrane proteins labeled with 5 nm
gold nanoparticles in live neutrons at video rates for several minutes
(Figs. 6e-g).
Discussion and outlook Many nanostructures exhibit multiple NLO signals which could provide
imaging contrast to track them. For example, GNR exhibits TPL, THG,
FWM, and photothermal signals. The rational choice for application
depends on the advantages and limitations of each modality as
well as the availability of instrument. Single beam modalities (e.g.,
multiphoton luminescence, THG, SHG) require relatively simple
instruments. Multiphoton luminescence and SHG can be performed on
a multiphoton microscope equipped with a fs Ti:sapphire laser, while
THG is often produced by longer excitation wavelengths (e.g., 1.2 μm)
produced by an optical parametric oscillator. More advanced
modalities (e.g., FWM, CARS, SRS, photothermal detection, transient
absorption) require a two-beam configuration which provides the
flexibility for better selectivity and/or higher sensitivity. Photothermal
microscopy is able to detect individual gold nanoparticles as small as
a few nm50,54,56. Compared to THG microscopy, FWM microscopy
provides more flexibility in tuning one or two laser wavelengths to an
electronic resonance within a nanomaterial. Such an advantage has
allowed FWM imaging of metallic nanotubes39. CARS and SRS provide
chemical selectivity based on Raman resonance, but at an imaging
speed higher than Raman microscopy by six orders of magnitude.
The transient absorption modality is suitable for mapping weakly
fluorescent molecules and nanostructures such as heme proteins111,112,
melanin107-110, and SWNTs49.
Strong intrinsic nonlinear optical (NLO) signals not only make
nanostructures promising agents for bio-imaging, but also advance
NLO microscopy for the study of interactions between nanomaterials
and live cells. Several approaches are promising to advance the
imaging of nano-bio interfaces. The first is the controllable synthesis
of advanced nanomaterials that generate strong NLO signals. Strong
NLO signals, small size, and low toxicity are desired for biological
applications. Studies that clarify how the NLO properties depend
on the size, shape, and chemical compositions of the nanostructure
are also of importance. The second direction is the development of
multimodal imaging platforms. In particular, the integration of atomic
force microscopy with NLO microscopy on a single platform should
provide a powerful tool to facilitate the study of NLO properties
of single nanostructures. The third direction is the application of
surface-modified nanostructures as biomarkers for NLO imaging
at the organism level. This would take advantage of the inherent
3D spatial resolution and relatively large penetration provided by
multiphoton microscopy. Besides the study of nano-bio interactions,
intrinsic NLO signals also provide great opportunities in the fields
of sensing, nanomanipulation (optical trapping, tweezers, and
surgery)126, optical data storing74, communication127, and energy. It
is expected that the exploration of NLO properties of nanomaterials,
although in its infancy, will generate an attractive and fast-growing
area in the near future, that is broadly applicable to biophotonics and
nanophotonics,
AcknowledgementsThis work was supported by NSF grant CBET0828832. The authors thank
Jackie Tyler for a careful reading of the manuscript.
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