Label-free imaging through nonlinear optical...

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]

MT146p264_273.indd 264 23/05/2011 10:46:21

mailto:[email protected]

Label-free imaging through nonlinear optical signals REVIEW

JUNE 2011 | VOLUME 14 | NUMBER 6 265

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).

MT146p264_273.indd 265 23/05/2011 10:46:24

REVIEW Label-free imaging through nonlinear optical signals

JUNE 2011 | VOLUME 14 | NUMBER 6266

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.

(a) (b) (c) (d)

(e) (f) (g) (h)

MT146p264_273.indd 266 23/05/2011 10:46:25



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.

(a) (c) (e) (g)

(b) (d) (f) (h)

MT146p264_273.indd 267 23/05/2011 10:46:27



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.

(a) (b) (c)

(d) (e) (f)

MT146p264_273.indd 268 23/05/2011 10:46:28



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

MT146p264_273.indd 269 23/05/2011 10:46:29



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.

(a) (b) (c)

(d) (e)

MT146p264_273.indd 270 23/05/2011 10:46:30



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)

MT146p264_273.indd 271 23/05/2011 10:46:32



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.

REFERENCES

1. Masters, B. R., and P. T. C. So, Handbook of Biomedical Nonlinear Optical Microscopy Oxford University Press: Oxford, New York, 2008.

2. Yue, S., et al., Laser Photonics Rev (2011), 1.

3. Eichelbaum, M., et al., Nanotechnology (2007) 18(35), 355702.

4. Farrer, R. A., et al., Nano Lett (2005) 5(6), 1139.

5. Okamoto, H., and Imura, K., J Mater Chem (2006) 16, 3920.

6. Wang, H., et al., Proc Natl Acad Sci USA (2005) 102, 15752.

7. Imura, K., et al., J Phys Chem B (2005) 109, 13214.

8. Park, J., et al., Opt Express (2008) 16, 1590.

9. Kim, H., et al., J Phys Chem C (2008) 112(33), 12721.

10. Wang, Q.-Q., et al., Nano Lett (2007) 7(3), 723.

11. Tong, L., et al., Angew Chem Int Ed (2010) 49(20), 3485.

12. Au, L., et al., ACS Nano (2010) 4, 35.

13. Johnson, J. C., et al., Nano Lett (2002) 2(4), 279.

14. Long, J. P., et al., Nano Lett (2007) 7(3), 831.

15. Nakayama, Y., et al., Nature (2007) 447(7148), 1098.

16. Butet, J., et al., Phys Rev Lett (2010) 105(7), 077401.

17. Butet, J., et al., Nano Lett (2010) 10(5), 1717.

18. Zavelani-Rossi, M., et al., Appl Phys Lett (2008) 92(9), 093119.

19. Le Xuan, L., et al., Small (2008) 4(9), 1332.

20. Bonacina, L., et al., Appl Phys B (2007) 87(3), 399.

Instrument citationFemtosecond Ti:Sapphire laser, Mira 900, Coherent Santa ClaraFemtosecond Ti: sapphire laser, Maitai HP, Spectra-Physics, FremontScanning confocal microscope, FV300_IX70, FV1000/IX81 & FV300/IX71, OlympusOptical parametric oscillator, OPO, Spectra-Physics, FremontPicosecond laser, Tsunami Spectra-PhysicsLock-in amplifier, SR844, Stanford Research Systems

MT146p264_273.indd 272 23/05/2011 10:46:33



21. Hsieh, C.-L., et al., Biomaterials (2010) 31(8), 2272.

22. Pantazis, P., et al., Proc Natl Acad Sci USA (2010) 107(33), 14535.

23. Zielinski, M., et al., Small (2009) 5(24), 2835.

24. Lippitz, M., et al., Nano Lett (2005) 5(4), 799.

25. Tai, S. P., et al., Adv Mater (2007) 19(24), 4520.

26. Liu, T.-M., et al., Appl Phys Lett (2006) 89(4), 043122.

27. Schwartz, O., and Oron, D., Nano Lett (2009) 9(12), 4093.

28. Jung, Y., et al., Nano Lett (2009) 9(6), 2440.

29. Chang, C.-F., et al., Opt Express (2010) 18(7), 7397.

30. Wang, Y., et al., Adv Opt Photon (2011) 3(1), 1.

31. Kim, H., et al., Nano Lett (2008) 8(8), 2373.

32. Masia, F., et al., Opt Lett (2009) 34(12), 1816.

33. Palomba, S., et al., J Optic Pure Appl Optic (2009) 11(11), 114030.

34. Jung, Y., et al., J Phys Chem C (2009) 113(7), 2657.

35. Zheng, Y., et al., Multichannel multiphoton imaging of metal oxides nanoparticles in biological system. In Multiphoton Microscopy in the Biomedical Sciences IV, 1 ed.; SPIE, San Jose, CA, USA (2004) 5323, 390.

36. Moger, J., et al., Opt Express (2008) 16(5), 3408.

37. Chen, H., et al., Opt Express (2009) 17(3), 1282.

38. Wagner, H. P., et al., J Crystal Growth (2000) 214-215, 747.

39. Kim, H., et al., Nano Lett (2009) 9(8), 2991.

40. Hendry, E., et al., Phys Rev Lett (2010) 105(9), 097401.

41. Staleva, H., and Hartland, G. V., J Phys Chem C (2008) 112(20), 7535.

42. Hartland, G. V., Chem Sci (2010) 1(3), 303.

43. Muskens, O. L., et al., Nano Lett (2006) 6(3), 552.

44. Zijlstra, P., et al., Nano Lett (2008) 8(10), 3493.

45. Tchebotareva, A. L., et al., ChemPhysChem (2009) 10(1), 111.

46. Burgin, J., et al., J Phys Chem C (2008) 112(30), 11231.

47. Carey, C. R., et al., J Phys Chem C (2009) 113(44), 19077.

48. Staleva, H., et al., Phys Chem Chem Phys (2009) 11(28), 5889.

49. Jung, Y., et al., Phys Rev Lett (2010) 105(21), 217401.

50. Gaiduk, A., et al., Chem Sci (2010) 1(3), 343.

51. Kulzer, F., et al., ChemPhysChem (2008) 9(12), 1761.

52. Blab, G. A., et al., Biophys J (2006) 90(1), L13.

53. Berciaud, S., et al., Phys Rev B (2006) 73(4), 045424.

54. Lasne, D., et al., Biophys J (2006) 91(12), 4598.

55. Berciaud, S., et al., Nano Lett (2005) 5(3), 515.

56. Cognet, L., et al., Proc Natl Acad Sci USA (2003) 100(20), 11350.

57. Boyer, D., et al., Science (2002) 297(5584), 1160.

58. Tcherniak, A., et al., Nano Lett (2010) 10(4), 1398.

59. Chang, W.-S., et al., Proc Natl Acad Sci USA (2010)



62. Mooradian, A., Phys Rev Lett (1969) 22, 185.

63. Mohamed, M. B., et al., Chem Phys Lett (2000) 317, 517.

64. Boyd, G. T., et al., Phys Rev B (1986) 33, 7923.

65. Bouhelier, A., et al., Phys Rev Lett (2005) 95, 267405.

66. Sonnichsen, C., and Alivisatos, A. P., Nano Lett (2004) 5(2), 301.

67. Lin, N., et al., J Phys Chem C (2011) 115(8), 3198.

68. Tong, L., et al., Photochem Photobiol (2009) 85, 21.

69. Tong, L., et al., Adv Mater (2007) 19, 3136.

70. Huff, T. B., et al., Langmuir (2007) 23, 1596.

71. Durr, N. J., et al., Nano Lett (2007) 7, 941.

72. Tong, L., et al., Langmuir (2009) 25(21), 12454.

73. Niidome, T., et al., J Control Release (2006) 114, 343.

74. Zijlstra, P., et al., Nature (2009) 459(7245), 410.

75. Li, Z., et al., Chin Phys Lett (2008) 25(5), 1776.

76. Campagnola, P. J., and Dong, C. Y., Laser Photonics Rev (2009) 5(1), 13.

77. Corn, R. M., and Higgins, D. A., Chem Rev (1994) 94, 107.

78. Campagnola, P. J., and Loew, L. M., Nat Biotech (2003) 21(11), 1356.

79. Mohler, W., et al., Methods (2003) 29(1), 97.

80. Finazzi, M., et al., Phys Rev B (2007) 76(12), 125414.

81. Kuo, T.-R., et al., Biomaterials (2009) 30(16), 3002.

82. Hsieh, C.-L., et al., Opt Express (2009) 17(4), 2880.

83. Boyd, R. W., Nonlinear Optics. 2nd ed.; Academic Press: Boston, 2003.

84. Cheng, J.-X., and Xie, X. S., J Opt Soc Am B (2002) 19(7), 1604.

85. Tsang, T. Y. F., Phys Rev A (1995) 52(5), 4116.

86. Barad, Y., et al., Appl Phys Lett (1997) 70(8), 922.

87. Yelin, D., and Silberberg, Y., Opt Express (1999) 5(8), 169.

88. Debarre, D., et al., Nat Meth (2006) 3(1), 47.

89. Olivier, N., et al., Science (2010) 329(5994), 967.

90. Chu, S.-W., et al., Opt Express (2003) 11(23), 3093.

91. Kim, E. M., et al., Phys Rev Lett (2005) 95(22), 227402.

92. Yelin, D., et al., Opt Express (2003) 11(12), 1385.

93. Andreas, V., J Phys D (2005) 38(5), R59.

94. Cheng, J.-X., and Xie, S. X., J Phys Chem B (2004) 108(3), 827.

95. Freudiger, C. W., et al., Science (2008) 322(5909), 1857.

96. Ploetz, E., et al., Appl Phys B (2007) 87(3), 389.

97. Nandakumar, P., and et al., New J Phys (2009) 11(3), 033026.

98. Potma, E. O., et al., Biophys J (2001) 80(6), 3019.

99. Evans, C. L., and Xie, X. S., Annu Rev Anal Chem (2008) 1(1), 883.

100. Le, T. T., et al., J Lipid Res (2010),

101. Wang, M. C., et al., Nat Meth (2011) 8(2), 135.

102. Slipchenko, M. N., et al., Analyst (2010) 135, 2613.

103. Saar, B. G., et al., Angew Chem Int Ed (2010) 122(32), 5608.

104. Tong, L., et al., J Phys Chem B (2007) 111(33), 9980.

105. Xu, P., et al., Mol Pharmaceutics (2008) 6(1), 190.

106. Ye, T., et al., Photochem Photobiol (2009) 85(3), 631.

107. Fu, D., et al., J Biomed Opt (2008) 13(5), 054036.

108. Fu, D., et al., Two-color excited-state absorption imaging of melanins. In Photonic Therapeutics and Diagnostics III, 1 ed.; SPIE, San Jose, CA, USA (2007) 6424, 642402.


110. Matthews, T. E., et al., Science Translational Medicine (2011) 3(71), 71ra15.

111. Fu, D., et al., Opt Lett (2007) 32(18), 2641.


113. Min, W., et al., Nature (2009) 461(7267), 1105.

114. van Dijk, M. A., et al., Phys Rev Lett (2005) 95(26), 267406.

115. Kang, S., et al., Nature Nanotech (2007) 2(4), 230.

116. Ellingson, R. J., et al., Phys Rev B (2005) 71(11), 115444.

117. Gaiduk, A., et al., Science (2010) 330(6002), 353.

118. Lasne, D., et al., Opt Express (2007) 15(21), 14184.

119. Lu, S., et al., Appl Phys Lett (2010) 96(11), 113701.

120. Cognet, L., et al., Anal Chem (2008) 80(7), 2288.

121. Berciaud, S., et al., Phys Rev Lett (2004) 93(25), 257402.

122. Mawatari, K., et al., Anal Chem (1998) 70(23), 5037.

123. Raduenz, R., et al., J Phys Chem A (2009) 113(9), 1674.

124. Paulo, P. M. R., et al., J Phys Chem C (2009) 113(27), 11451.

125. Octeau, V., et al., ACS Nano (2009) 3(2), 345.

126. Mathew, M., et al., Rev Sci Instrum (2009) 80(7), 073701.

127. Lee, L., et al., A Novel Nano-Photonic Quantum Dots Optical Fiber (NQDOF) for future photonic communications In IEEE-Nano 2007 (2007) 480.

MT146p264_273.indd 273 23/05/2011 10:46:34

Label-free imaging through nonlinear optical...

Documents

Transcript of Label-free imaging through nonlinear optical...