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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 16, Iss. 13 — Jun. 23, 2008
  • pp: 9534–9548
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Cell tracking and detection of molecular expression in live cells using lipid-enclosed CdSe quantum dots as contrast agents for epi-third harmonic generation microscopy

Chieh-Feng Chang, Chao-Yu Chen, Fu-Hsiung Chang, Shih-Peng Tai, Cheng-Ying Chen, Che-Hang Yu, Yi-Bing Tseng, Tsung-Han Tsai, I-Shuo Liu, Wei-Fang Su , and Chi-Kuang Sun  »View Author Affiliations


Optics Express, Vol. 16, Issue 13, pp. 9534-9548 (2008)
http://dx.doi.org/10.1364/OE.16.009534


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Abstract

We demonstrated that lipid-enclosed CdSe quantum dots (LEQDs) can function as versatile contrast agents in epi-detection third harmonic generation (THG) microscopy for biological applications in vivo. With epi-THG intensities 20 times stronger than corresponding fluorescence intensities from the same LEQDs under the same conditions of energy absorption, such high brightness LEQDs were proved for the abilities of cell tracking and detection of specific molecular expression in live cancer cells. Using nude mice as an animal model, the distribution of LEQD-loaded tumor cells deep in subcutaneous tissues were imaged with high THG contrast. This is the first demonstration that THG contrast can be manipulated in vivo with nanoparticles. By linking LEQDs with anti-Her2 antibodies, the expression of Her2/neu receptors in live breast cancer cells could also be easily detected through THG. Compared with fluorescence modalities, the THG modality also provides the advantage of no photobleaching and photoblinking effects. Combined with a high penetration 1230 nm laser, these novel features make LEQDs excellent THG contrast agents for in vivo deep-tissue imaging in the future.

© 2008 Optical Society of America

1. Introduction

In this paper, we developed lipid-enclosed quantum dots (LEQDs) that are functional under different excitation sources to act as efficient THG contrast agents for contrast manipulation and for molecular imaging. Due to coherent effects, the epi-THG intensity from an LEQD was found to nonlinearly increase with the particle size and would reach saturation when the particle diameter exceeded 100 nm, beginning from which destructive interference from different parts of a large nanoparticle would occur. Enhanced by three-photon resonance, the detected epi-THG intensities of ~130 nm LEQDs were found to be 20 times stronger than the corresponding fluorescence intensities from the same LEQDs under the same conditions of energy absorption. We then demonstrated that such high-THG-brightness LEQDs could be used to label live cells for in vivo cell tracking and for detection of specific molecular expression. Using nude mice as an animal model, CT-26 tumor cells loaded with LEQD could be clearly imaged in vivo by epi-THG microscopy deep in subcutaneous tissues. We also performed experiments on cultured human breast cancer cells and proved that the abilities of high-brightness LEQDs could be further extended to the detection of specific molecular expression. By linking LEQDs with anti-Her2 antibodies, AU565 cells with high Her2/neu receptor expression on cell surfaces could be selectively identified from other cell lines with low expression. These experiments indicated that through the THG mechanism LEQDs can provide even brighter signals than fluorescence under the same condition of energy deposition, which enables higher cell viability and a much improved penetration depth. As a further step from the manipulation of THG contrast we previously reported in fixed cells, ex vivo, or using acetic acid [10

10. C.-H. Yu, S.-P. Tai, C.-T. Kung, I.-J. Wang, H.-C. Yu, H.-J. Huang, W.-J. Lee, Y.-F. Chan, and C.-K. Sun, “In vivo and ex vivo imaging of intra-tissue elastic fibers using third-harmonic-generation microscopy,” Opt. Express 15, 11167–11177 (2007), http://www.opticsinfobase.org/abstract.cfm?URI=oe-15-18-11167. [CrossRef] [PubMed]

, 27

27. S.-P. Tai, Y. Wu, D.-B. Shieh, L.-J. Chen, K.-J. Lin, C.-H. Yu, S.-W. Chu, C.-H. Chang, X.-Y. Shi, Y.-C. Wen, K.-H. Lin, T.-M. Liu, and C.-K. Sun, “Molecular Imaging of Cancer Cells Using Plasmon-Resonant-Enhanced Third-Harmonic-Generation in Silver Nanoparticles,” Adv. Mater. 19, 4520–4523 (2007). [CrossRef]

, 35

35. C.-H. Yu, S.-P. Tai, C.-T. Kung, W.-J. Lee, Y.-F. Chan, H.-L. Liu, J.-Y. Lyu, and C.-K. Sun, “Molecular third-harmonic-generation microscopy through resonance enhancement with absorbing dye,” Opt. Lett. 33, 387–389 (2008). [CrossRef] [PubMed]

], these results demonstrated for the first time that THG contrast can be manipulated in vivo using nanoparticles in live animals. Combined with the high-penetration and high-viability capabilities of the excitation laser [3

3. C.-K. Sun, S.-W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C.-Y. Lin, and H.-J. Tsai, “Higher harmonic generation microscopy for developmental biology,” J. Struct. Biol. 147, 19–30 (2004). [CrossRef] [PubMed]

], these unique features make LEQDs excellent THG contrast agents for future microscopy applications in vivo, especially when deep-tissue biomolecular imaging or contrast manipulation is required.

2. Materials and methods

2.1 Synthesis of lipid-enclosed CdSe quantum dots

Fig. 1. (a) Absorption and emission spectra of ~5 nm CdSe QDs. Black line: absorption; red line: emission. (b) Transmission electron micrograph of ~100 nm lipid-enclosed CdSe QDs. Scale bar: 10 nm

2.2 Cell cultures

Human cervical cancer cells (HeLa), two human breast cancer cell lines (AU565 and MCF7) and mouse colorectal adenocarcinoma cells (CT-26) were obtained from the American Type Culture Collections (ATCC) (MD, USA). HeLa, MCF7 and AU565 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, whereas CT-26 cells were cultured in Roswell Park Memorial Institute (RPMI) medium plus 10% serum. Routine cell culture was carried out at 37 °C with supplementation of 5% CO2.

2.3 Procedures for loading LEQDs into HeLa and CT-26 cells

HeLa and CT-26 cells (2×104-1×105) were seeded in each of the 35 mm culture plate wells for 24 hours before loading of LEQDs. Cationic LEQDs of mass 5 µg were incubated with cells in 1.5 ml culture medium at 37 °C for overnight. After brief washing, these cells were either fixed for THG observation or imaged when still living. Due to the cationic surface charge, the loading efficiency of LEQDs to cells was high and therefore the total amount of LEQDs used to load cells was low. In this case nearly all cells (95%) were loaded with LEQDs, which was verified using flow cytometry analysis. Electron microscope pictures also confirmed that cationic lipids help nanoparticles escape from endosomes and stay in the peri-nuclear region.

2.4 Tumor model for THG imaging

Around 5×105 of LEQD-loaded CT-26 cells were injected into the dermis tissues of nude mice (Narl: ICR-Foxn1nu) to initiate tumors. After one week, in vivo epi-THG images were taken from anesthetized animals. The experimental protocols were approved by the National Taiwan University Institutional Animal Care and Use Committee (NTU-IACUC) and by the National Taiwan University Hospital Institutional Animal Care and Use Committee (NTUH-IACUC).

2.5 Synthesis of anti-Her2 antibody- linked LEQDs

To assemble receptor-targeting LEQDs, protein A-streptavidin (PAST) fusion protein was used as a bifunctional adaptor. The genetically engineered PAST fusion protein was expressed in bacteria, harvested, and then isolated homogeneously by an affinity column. Neutral LEQDs were coated with PAST through biotin linkage. Anti-Her2 antibodies (Herceptin, Genentech Co., CA, USA) were further linked to LEQD surfaces via protein A interaction.

2.6 Cell labeling with anti-Her2-linked LEQDs

AU565 and MCF7 cells (2×104-1×105) were seeded in each of the 35 mm culture plate wells for 24 hours before QD labeling. Anti-Her2-linked LEQDs (1.25 µg) were diluted in 1.5 ml of basal medium, and were subsequently added to each of the culture wells. After incubation for 1 hour, cells were washed with phosphate buffered saline (PBS), replaced with complete medium, and further incubated at 37 °C with the supplementation of 5% CO2 for another 24 hours before imaging.

2.7 Epi-THG/MPEF microscope

One optical platform with simultaneous epi (reflection) type THG and multiphoton excited fluorescence (MPEF) microscope was developed and adopted in this study [13

13. S.-W. Chu, I.-H. Chen, T.-M. Liu, C.-K. Sun, S.-P. Lee, B.-L. Lin, P.-C. Cheng, M.-X. Kuo, D.-J. Lin, and H.-L. Liu, “Nonlinear bio-photonic crystal effects revealed with multimodal nonlinear microscopy,” J. Microsc. 208, 190–200 (2002). [CrossRef] [PubMed]

]. The study of THG microscopy using LEQDs as contrast agents was performed using a home-built femtosecond Cr:forsterite laser centered at 1230 nm with a 130 fs pulsewidth and a 110 MHz repetition rate [8

8. S.-P. Tai, W.-J. Lee, D.-B. Shieh, P.-C. Wu, H.-Y. Huang, C.-H. Yu, and C.-K. Sun, “In vivo optical biopsy of hamster oral cavity with epi-third-harmonic-generation microscopy,” Opt. Express 14, 6178–6187 (2006), http://www.opticsinfobase.org/abstract.cfm?URI=oe-14-13-6178. [CrossRef] [PubMed]

, 15

15. S.-P. Tai, T.-H. Tsai, W.-J. Lee, D.-B. Shieh, Y.-H. Liao, H.-Y. Huang, K. Zhang, H.-L. Liu, and C.-K. Sun, “Optical biopsy of fixed human skin with backward-collected optical harmonics signals,” Opt. Express 13, 8231–8242 (2005), http://www.opticsinfobase.org/abstract.cfm?URI=oe-13-20-8231 . [CrossRef] [PubMed]

]. The spectral full width at half maximum of the laser output was about 20 nm. The infrared laser beam was first shaped by a telescope, and then directed into a modified beam-scanning system (Olympus FV300) and an upright microscope (Olympus BX51). When the observed cell specimens were placed on glass slides, an infrared (IR) waterimmersion objective with a high numerical aperture (NA) and a short working distance (WD) (Olympus UplanApo/IR 60X/NA 1.2/WD 0.2 mm) was used to increase the spatial resolution and the THG efficiency. For in vivo animal studies, an IR water-immersion objective with a 2 mm working distance (Olympus LUMplanFL/IR 60X/NA 0.9/WD 2 mm) was used to perform deep-tissue observation. After anaesthetization, the nude mouse was put under our epi-THG microscope as shown in Fig. 2(b) with an electrical blanket to maintain the body temperature of the test animal. The epi-THG signals were collected using the same objective for illumination; details of the epi-THG microscope can be found in [8

8. S.-P. Tai, W.-J. Lee, D.-B. Shieh, P.-C. Wu, H.-Y. Huang, C.-H. Yu, and C.-K. Sun, “In vivo optical biopsy of hamster oral cavity with epi-third-harmonic-generation microscopy,” Opt. Express 14, 6178–6187 (2006), http://www.opticsinfobase.org/abstract.cfm?URI=oe-14-13-6178. [CrossRef] [PubMed]

]. To ensure the signals we collected were THG, a spectrometer was used to confirm the detected wavelength before imaging and our power-dependent study also verified the cubic dependency of the signals.

Fig. 2. (a) The tumor region in the dorsal skin was restrained on our home-made device for observation. (b) After anesthetization, the nude mouse was put under our epi-THG microscope with an electric blanket to maintain the body temperature.

2.8 Numerical simulations

Numerical simulations were performed to verify the origin of the detected THG signals. The dyadic Green’s function was used to calculate the THG strength both in the epi- and the forward-directions [40

40. J. X. Cheng and X. S. Xie, “Green’s function formulation for third-harmonic generation microscopy,” J. Opt. Soc. Am. B: Opt. Phys. 19, 1604–1610 (2002). [CrossRef]

]:

E(3ω)(R)VdV(I¯+k32)exp(ik3R·r)R·P(3ω)(r),
(1)
P(3ω)θdθ02πdϕE(3ω)(R)2R2sinθ,
(2)

where R and r are coordinates of observation and source points, respectively, Ī is the idemfactor, ∇ is the dyadic del operator, k3 is the wave-vector amplitude at 3ω in the matrix, and P (3ω)(r) is the laser-induced third-order polarization inside the particle. The value of k3 is calculated by k 3=n 3·2π/λ, where n3=1.344 is the refractive index of water in this case [41

41. M. J. Weber, Handbook of Optical Materials (CRC Press, 2003).

]. To calculate P (3ω)(r), the excitation field was modeled as a focused Gaussian beam, and the refractive index of CdSe was calculated with the associated Sellmeier equation [41

41. M. J. Weber, Handbook of Optical Materials (CRC Press, 2003).

] to determine the medium-dependent optical distance z inside the particle [42

42. J. M. Schins, T. Schrama, J. Squier, G. J. Brakenhoff, and M. Muller, “Determination of material properties by use of third-harmonic generation microscopy,” J. Opt. Soc. m. B: Opt. Phys. 19, 1627–1634 (2002). [CrossRef]

]. The value of 2.523 is used as the refractive index of CdSe at 1230 nm [41

41. M. J. Weber, Handbook of Optical Materials (CRC Press, 2003).

]. Because the particle sizes were much smaller than the wavelength, diffraction at the particle-solvent interface was ignored in our calculation. The observation cone angle was defined as θ=115° to 180° and 0° to 65° for epi-THG and forward-THG (F-THG) calculations, respectively, corresponding to the NA 1.2 objective used in the experiment. The value was calculated by α=sin-1(NA/n 1)=65°, where n 1 is the refractive index at ω in the matrix [40

40. J. X. Cheng and X. S. Xie, “Green’s function formulation for third-harmonic generation microscopy,” J. Opt. Soc. Am. B: Opt. Phys. 19, 1604–1610 (2002). [CrossRef]

] and is chosen to be 1.325 for water here [41

41. M. J. Weber, Handbook of Optical Materials (CRC Press, 2003).

]. The volume of integration V was limited to the particle region assuming that the χ(3) value of CdSe is much larger than that of the solvent [43

43. J. T. Seo, S. M. Ma, Q. Yang, L. Creekmore, R. Battle, H. Brown, A. Jackson, T. Skyles, B. Tabibi, and W. Yu, “Large Resonant Third-order Optical Nonlinearity of CdSe Nanocrystal Quantum Dots,” J. Phys.: Conf. Ser. 38, 91–94 (2006). [CrossRef]

]. To take into consideration the polydispersity of particles, the calculated curves were furthermore weighted by a Gaussianshaped size distribution, with a standard deviation proportional to the particle diameter:

Ppoly(3ω)(d)dddP(3ω)(d)·1σexp((dd)22σ2),
(3)

The standard deviation is calculated by σ=(d′/d refσ ref, where the referenced standard deviation of size distribution is derived from TEM statistics and is σ ref=30 nm for d ref=130 nm.

3. Results and discussion

3.1 Epi-THG characteristics of lipid-enclosed CdSe QDs

I(d)[1ff(1512×512m=1512n=1512S(f,m,n))]·y(V)·d3,
(4)

where f is the number of frames in the image, S(f,m,n) is the pixel intensities of the image, y(V) is the experimentally-determined weighting factor of the PMT output as a function of the supplied voltage V, and d is the measured particle size. The simulation results were then fitted to the experimental data by

I(d)C·Ppoly(3ω)(d),
(5)

where C is a fitting constant accounting for the effective χ(3) inside LEQDs, all reflection/absorption losses of signals, and all constant terms dropped in Eqs. (1)-(4) to speed up calculation. Since a common value of C is used to fit all data of different LEQD diameters, the fitting process was performed to address the size dependence instead of the resonance effect. The measured relative epi-THG intensity of one single LEQD, lipid nanoparticle (GEC-Chol/Chol, 1:1) and CdSe QD is then plotted in Fig. 3(j) as a function of particle diameter, along with the simulation results on direct-epi- and forward-THG from LEQDs, with C as the only fitting constant. Using the sampling model aforementioned, numerical simulations were performed by positioning the particle at different locations inside the confocal volume, and the calculated THG intensities were then added. The calculated curve in this way deviated by less than 5% from the curve assuming a fixed particle at the center of the focus when normalized to the same scale, so the latter fixed-position approach could also be used to expedite the calculation. Due to the coherent THG process, when the particle size is much smaller than the THG wavelength, the generated THG field intensities from different parts of the nanoparticle constructively interfere with each other, causing the ∝d6 power increase with the particle size for both forward and backward THG [44

44. D. Debarre, N. Olivier, and E. Beaurepaire, “Signal epidetection in third-harmonic generation microscopy of turbid media,” Opt. Express 15, 8913–8924 (2007), http://www.opticsinfobase.org/abstract.cfm?URI=oe-15-14-8913. [CrossRef] [PubMed]

], as has been reflected by the simulation curves shown in Fig. 3(j). However, when the particle size increases to a fraction of the THG wavelength (100 nm in our case), the epi-collected THG field intensities generated from different parts of the nanoparticle will start to show phase variations, due to different excitation time and due to different distances from the detector, thus starting to cause destructive interference. This coherent saturation effect limits the optimized particle size for epi-THG detection, which is slightly larger than Δx≈ℏ/Δp due to the 3D shape of the nanoparticle, where Δp is the momentum mismatch between three incoming 1230 nm photons and one epi-detected THG photon.

In addition to the curves assuming monodisperse LEQDs, which are shown as cyan and dark yellow dashed lines in the figure, curves assuming a Gaussian-shaped particle-size distribution are also plotted as solid lines. As can be seen in the figure, the experimental data fit very well to the simulation on direct-epi-THG with polydispersity (black solid line) but deviate obviously from the trace of the forward-THG (red solid line), supporting the proposed coherent saturation effect. With a 1230 nm excitation light, the optimized nanoparticle size for epi-THG detection is thus around 100 nm. In the following reported imaging experiments, we used LEQDs 130 nm in diameter for efficient THG generation. This size-dependent experiment also suggested that the signals we measured were direct-epi-THG, not forward-THG back-scattered after generation.

Fig. 3. Ep-THG intensities of nanoparticles with different sizes and materials. Figures (a)-(i) show the epi-THG images with different materials in solution: (a) Deionized water; (b) 10 nm GEC lipid nanoparticles; (c) 50 nm GEC lipid nanoparticles; (d) 100 nm GEC lipid nanoparticles; (e) 200 nm GEC lipid nanoparticles; (f) 100 nm DPPC lipid nanoparticles; (g) 5 nm CdSe QDs; (h) 30 nm GEC-lipid-enclosed CdSe QDs; (i) 130 nm GEC-lipid-enclosed CdSe QDs; (j) Epi-THG power vs. diameter for GEC-lipid-enclosed CdSe QDs (black circle), GEC lipid nanoparticles (green square), and ~5 nm CdSe QDs (blue triangle). Cyan and dark yellow dashed lines represent epi-THG and F-THG, respectively, from monodisperse LEQDs; black and red lines represent epi-THG and F-THG, respectively, assuming a Gaussian distribution in polydispersity. Epi-THG intensities from CdSe QDs and GEC lipid nanoparticles are much weaker than those from GEC-lipid-enclosed CdSe QDs.

The influence of the multiple-interface effect inside LEQDs is discussed here. The particles used in our experiments had a maximum diameter of ~130 nm, which is much smaller than the excitation wavelength of 1230 nm. Since each LEQD contained more than tens of 5 nm CdSe quantum dots, the spacing between QDs inside the sphere would be on the order of 10 nm or less. As a result, the multiple-interface effect should not be a dominant factor in the observed strong THG from LEQDs, because such an effect is the result of the Gouy phase shift effect and is obvious only when interfaces are separated by a distance comparable to the confocal parameter. Inside our LEQDs, THG field intensities generated from these closely-packed QDs would have a coherent phase relationship determined by their separation distance, rather than experiencing different phase shifts due to the Gouy effect, which is the cause of the usually called interface effect. To verify this concept, we performed additional simulations for verification. The sample structure used in the simulation consisted of two thin slabs 10 nm in thickness, separated by a distance ranging from 0 to 100 nm, as shown in Fig. 4(a). The χ(3) value of the surrounding matrix was assumed to be much smaller than that of the slab, so THG from the matrix was ignored. The focal position of the incident Gaussian beam was scanned across the entire sample structure, and the maximum value in each scan was recorded. Because of the nature of this layered structure, a phase correction term has to be added to the excitation beam in the second slab region, which can be derived as

Δϕ=2πλ(n1,slab2n1,mediumn1,medium)·δ,
(6)

where δ is the separation distance between two slabs. The wavelength was set to 1230 nm, NA to 1.2, cone angle to 65°, and material parameters of CdSe and water were used for the slab and the medium regions, respectively, as described in Section 2.8. The result is plotted in Fig. 4(b). As can be seen from the figure, when the slab thickness and spacing between them are much smaller than the wavelength, increasing the total number of interfaces does not necessarily lead to stronger THG signals. It is the total volume and the coherent effect that determined the measured THG intensity. This is especially obvious in epi-THG, because destructive interference can cause significant signal reduction at specific intervals of slab separation.

Fig. 4. (a) Schematic of the structure used in simulations to illustrate the coherent versus the multiple-interface effects. The thickness of each slab is 10 nm, while the separation between them is 0-100 nm. (b) Simulation results on the intensities of forward- and epi-THG from the structure. Black line: forward-THG; red line: epi-THG.

3.2 Imaging LEQDs in cells using epi-THG microscopy vs. MPEF microscopy

QDs are efficient fluorophores for confocal and MPEF microscopy techniques. They can thus provide fluorescence signals to reflect the degree of absorption due to the effect of three-photon-resonance enhancement, which causes THG contrast agents to absorb the excitation light through three-photon absorption or through generated third-harmonic waves [45

45. G. Veres, S. Matsumoto, Y. Nabekawa, and K. Midorikawa, “Enhancement of third-harmonic generation in absorbing media,” Appl. Phys. Lett. 81, 3714–3716 (2002). [CrossRef]

]. To quantitatively compare signal intensities of THG and fluorescence from LEQDs, HeLa cells were loaded with LEQDs and then imaged with our microscope, which simultaneously recorded THG and MPEF images. As mentioned in Section 2.1, one advantage of using LEQDs is that we can choose different lipids to synthesize various kinds of LEQDs for different applications, adding versatility and flexibility to this technique. As a result, the composition of cationic cholesterol (GEC-Chol) and standard cholesterol in a 1:1 molar ratio was used to facilitate cell uptake because cationic LEQDs could be efficiently absorbed by cells due to additional positive charge on their surfaces. Figure 5(a) shows one measured epidirection spectrum of the LEQDs excited by our fs Cr:forsterite laser. Strong epi-THG signals, >20 times larger than the three-photon fluorescence (3PF) signals around 580 nm, could be detected and there was no crosstalk between THG and 3PF. This result is twofold in significance. From one point of view, our measurement verified that with the same signal intensities, the light absorption in contrast agents due to the effect of real-state-resonance enhancement in epi-THG microscopy is >20X less than the corresponding fluorescence-based technologies. From the other, with the same amount of energy deposition into contrast agents, epi-THG in LEQDs can provide much brighter signals over fluorescence-based modalities, not to mention the adopted laser can also offer much reduced energy deposition into surrounding biotissues. Compared with the fluorescence microscopy using quantum dots, THG microscopy using LEQDs can therefore simultaneously yield a better signal-to-noise ratio (SNR) and result in much reduced photodamage. Furthermore, unlike fluorescence from QDs, no photoblinking or photobleaching was observed when these LEQDs were imaged using epi-THG microscopy, which provides another advantage of this virtual-transition-based technique. To visually verify this result, Fig. 5(b) shows a typical epi-THG image of LEQDloaded HeLa cells, in which bright spots can be clearly seen in the cytosol region. Figure 5(c) shows the simultaneously acquired 3PF image after normalization, with the 3PF wavelength corresponding to the QD fluorescence wavelength. Because the THG intensities were much stronger than 3PF, Fig. 5(b) contained most of the bright spots in (c) and revealed more information than fluorescence. Under the experimental condition with a low PMT voltage, the possibility was excluded that these extra spots were from intrinsic cell structures. The result in Fig. 5(b) is then significant in several ways for LEQDs to be eligible contrast agents for epi- THG microscopy in vivo. First, the epi-THG signals were strong in Fig. 5(b), indicating that LEQDs were efficient epi-THG generators in cells. Second, because the HeLa cells were washed before imaging (see Section 2.3), dead cells and particles suspending in solution would be carried away by the procedure; for THG to be observed from LEQDs, these particles must be able to penetrate into cells on one hand and have no immediate toxicity on the other. For further verification, those cells were cultured for additional 48 hours after washing and their viability was confirmed using MTT assay. From the experimental results, it can be concluded that these LEQDs can be effectively absorbed by cells, pose no immediate toxicity to them, and can be efficiently observed through epi-THG. These features are critical in choosing contrasts agents for microscopy in vivo, and the LEQDs we developed clearly meet these criteria and are suitable for applications like cell tracking.

Fig. 5. One typical measured backward emission spectrum of these LEQDs excited by our fs Cr:fosterite laser is shown as (a). The observed epi-THG was ~20X stronger than the 3PF from the LEQDs. The cationic LEQDs in HeLa cells were also imaged by (b) THG microscopy and (c) 3PF microscopy. Scale bar: 20 µm.

3.3 In vivo tumor cell tracking by epi-THG microscopy

Fig. 6. (a) (539 kB) An example movie shows a stack of depth-resolved in vivo horizontal sections from epi-harmonic generation microscopy in normal subcutaneous tissues of a nude mouse. Depth range: 70-100 µm. (b) (1.76 MB) An example movie shows a stack of depthresolved in vivo horizontal sections in the subcutaneous tissues in the tumor-induced region loaded with LEQD. The red color represents epi-THG and the green epi-SHG. Depth range: 70-120 µm. Image size: 120×120 µm. [Media 1][Media 2]

3.4 Epi-THG Detection of Her2 Expression in live breast cancer cells

Fig. 7. Demonstration of THG imaging using anti-Her2-linked neutral LEQDs as contrast agents to identify the Her2 expression in live breast cancer cells with normal (MCF7) and high (AU565) expression. (a) Schematic of anti-Her2-linked LEQDs (b) THG image of AU565 cells not applied with LEQDs. (c) THG image of AU565 cells applied with LEQDs. (d) THG image of AU565 cells applied with anti-Her2-linked LEQDs. (e) THG image of MCF7 cells not applied with LEQDs. (f) THG image of MCF7 cells applied with LEQDs. (f) THG image of MCF7 cells applied with anti-Her2-linked LEQDs. Scale bar: 30 µm. Rich bright THG spots can only be observed in the AU565 cells applied with anti-Her2-linked LEQDs, and thus our developed THG microscopy can identify the Her2 expression levels in live breast cancer cells with normal and overexpressing Her2 receptors.

4. Conclusion

In this paper, we developed lipid-enclosed CdSe quantum dots as a new type of contrast agents in THG microscopy. They are efficient THG generators, 20X brighter than the silver nanoparticles we previously reported. They are potentially more biocompatible than pure quantum dots and possess no immediate biohazard in nude mice and cultured human cells. By injection of LEQD-loaded cancer cells into nude mice, the tumor region could be clearly imaged with epi-THG microscopy in vivo after one week, even deep in subcutaneous tissues. By linking LEQDs with anti-Her2 antibodies, the expression levels of Her2/neu receptors in two live cell lines of human breast cancer could be imaged and distinguished by our epi-THG microscope.

Compared with fluorescence microscopy using quantum dots, as THG contrast agents these LEQDs have no photobleaching or photoblinking problems, which allows long-term tracing of cell positions or molecular expression. In addition, even with an epi-collection scheme our developed LEQDs can generate THG signals 20X stronger than fluorescence, thus achieving a higher SNR and better penetration capability when the energy deposited in quantum dots are kept the same. If fluorescence signals are to reach the same intensities as THG, then more energy has to be stored in quantum dots for more fluorescence relaxation. Compared with the 1230 nm laser used in our experiments, in confocal or two-photon fluorescence microscopy a shorter wavelength is typically used for excitation, which reduces the penetration depth and may bring more photodamage to biological specimens. Combining a 1230 nm laser and the LEQDs we demonstrated, deeper penetration and higher cell survivability can be achieved, which in turn enables long-time observation on a grander time scale. Bright, biocompatible and versatile, the developed LEQDs prove to be a competent type of contrast agents in THG microscopy in vivo. Our study suggests that these LEQDs can be used in various types of biological applications in the future.

Acknowledgments

The authors gratefully acknowledge financial support from the National Health Research Institute (NHRI-EX97-9201EI), National Science Council (NSC96-2120-M-002-014; NSC96-2320-B-002-086), National Taiwan University Research Center for Medical Excellence-Division of Genomic Medicine, and Frontier Research of National Taiwan University.

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D. Debarre, W. Supatto, E. Farge, B. Moulia, M. C. Schanne-Klein, and E. Beaurepaire, “Velocimetric third-harmonic generation microscopy: micrometer-scale quantification of morphogenetic movements in unstained embryos,” Opt. Lett. 29, 2881–2883 (2004). [CrossRef]

5.

W. Supatto, D. Debarre, B. Moulia, E. Brouzes, J. L. Martin, E. Farge, and E. Beaurepaire, “In vivo modulation of morphogenetic movements in Drosophila embryos with femtosecond laser pulses,” Proc. Nat. Acad. Sci. U.S.A. 102, 1047–1052 (2005). [CrossRef]

6.

D. Debarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods 3, 47–53 (2006). [CrossRef]

7.

T.-H. Tsai, C.-Y. Lin, H.-J. Tsai, S.-Y. Chen, S.-P. Tai, K.-H. Lin, and C.-K. Sun, “Biomolecular imaging based on far-red fluorescent protein with a high two-photon excitation action cross section,” Opt. Lett. 31, 930–932 (2006). [CrossRef] [PubMed]

8.

S.-P. Tai, W.-J. Lee, D.-B. Shieh, P.-C. Wu, H.-Y. Huang, C.-H. Yu, and C.-K. Sun, “In vivo optical biopsy of hamster oral cavity with epi-third-harmonic-generation microscopy,” Opt. Express 14, 6178–6187 (2006), http://www.opticsinfobase.org/abstract.cfm?URI=oe-14-13-6178. [CrossRef] [PubMed]

9.

S.-Y. Chen, C.-S. Hsieh, S.-W. Chu, C.-Y. Lin, C.-Y. Ko, Y.-C. Chen, H.-J. Tsai, C.-H. Hu, and C.-K. Sun, “Noninvasive harmonics optical microscopy for long-term observation of embryonic nervous system development in vivo,” J. Biomed. Opt. 11, 054022 (2006). [CrossRef] [PubMed]

10.

C.-H. Yu, S.-P. Tai, C.-T. Kung, I.-J. Wang, H.-C. Yu, H.-J. Huang, W.-J. Lee, Y.-F. Chan, and C.-K. Sun, “In vivo and ex vivo imaging of intra-tissue elastic fibers using third-harmonic-generation microscopy,” Opt. Express 15, 11167–11177 (2007), http://www.opticsinfobase.org/abstract.cfm?URI=oe-15-18-11167. [CrossRef] [PubMed]

11.

E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, “In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy,” J. Microsc. 229, 141–150 (2008). [CrossRef] [PubMed]

12.

V. Barzda, C. Greenhalgh, J. Aus der Au, S. Elmore, J. van Beek, and J. Squier, “Visualization of mitochondria in cardiomyocytes by simultaneous harmonic generation and fluorescence microscopy,” Opt. Express 13, 8263–8276 (2005), http://www.opticsinfobase.org/abstract.cfm?URI=oe-13-20-8263. [CrossRef] [PubMed]

13.

S.-W. Chu, I.-H. Chen, T.-M. Liu, C.-K. Sun, S.-P. Lee, B.-L. Lin, P.-C. Cheng, M.-X. Kuo, D.-J. Lin, and H.-L. Liu, “Nonlinear bio-photonic crystal effects revealed with multimodal nonlinear microscopy,” J. Microsc. 208, 190–200 (2002). [CrossRef] [PubMed]

14.

C.-K. Sun, C.-C. Chen, S.-W. Chu, T.-H. Tsai, Y.-C. Chen, and B.-L. Lin, “Multiharmonic-generation biopsy of skin,” Opt. Lett. 28, 2488–2490 (2003). [CrossRef] [PubMed]

15.

S.-P. Tai, T.-H. Tsai, W.-J. Lee, D.-B. Shieh, Y.-H. Liao, H.-Y. Huang, K. Zhang, H.-L. Liu, and C.-K. Sun, “Optical biopsy of fixed human skin with backward-collected optical harmonics signals,” Opt. Express 13, 8231–8242 (2005), http://www.opticsinfobase.org/abstract.cfm?URI=oe-13-20-8231 . [CrossRef] [PubMed]

16.

R. R. Anderson and J. A. Parrish, “The Optics of Human Skin,” J. Invest. Dermatol. 77, 13–19 (1981). [CrossRef] [PubMed]

17.

C.-K. Sun, Series in Advances in Biochemical Engineering/Biotechnology, Special Volume 95: Microscopy Techniques (Springer-Verlag, Berlin, 2005), Chap. “Higher harmonic generation microscopy.”

18.

S.-W. Chu, S.-P. Tai, C.-L. Ho, C.-H. Lin, and C.-K. Sun, “High-resolution simultaneous three-photon fluorescence and third-harmonic-generation microscopy,” Microsc. Res. Tech 66, 193–197 (2005). [CrossRef] [PubMed]

19.

G. O. Clay, A. C. Millard, C. B. Schaffer, J. Aus-der-Au, P. S. Tsai, J. A. Squier, and D. Kleinfeld, “Spectroscopy of third-harmonic generation: evidence for resonances in model compounds and ligated hemoglobin,” J. Opt. Soc. Am. B: Opt. Phys. 23, 932–950 (2006). [CrossRef]

20.

R. D. Schaller, J. C. Johnson, and R. J. Saykally, “Nonlinear Chemical Imaging Microscopy: Near-Field Third Harmonic Generation Imaging of Human Red Blood Cells,” Anal. Chem 72, 5361–5364 (2000). [CrossRef] [PubMed]

21.

H. Kano and S. Kawata, “Two-photon-excited fluorescence enhanced by a surface plasmon,” Opt. Lett. 21, 1848–1850 (1996). [CrossRef] [PubMed]

22.

K. Kneipp, H. Kneipp, I. Itzkan, R. R. Dasari, and M. S. Feld, “Ultrasensitive chemical analysis by Raman spectroscopy,” Chem. Rev. 99, 2957–2976 (1999). [CrossRef]

23.

B. Lamprecht, J. R. Krenn, A. Leitner, and F. R. Aussenegg, “Resonant and Off-Resonant Light-Driven Plasmons in Metal Nanoparticles Studied by Femtosecond-Resolution Third-Harmonic Generation,” Phys. Rev. Lett. 83, 4421–4424 (1999). [CrossRef]

24.

T.-M. Liu, S.-P. Tai, C.-H. Yu, Y.-C. Wen, S.-W. Chu, L.-J. Chen, M. R. Prasad, K.-J. Lin, and C.-K. un, “Measuring plasmon-resonance enhanced third-harmonic χ(3) of Ag nanoparticles,” Appl. Phys. Lett. 89, 043122 (2006). [CrossRef]

25.

S. Nie and S. R. Emory, “Probing Single Molecules and Single Nanoparticles by Surface-Enhanced Raman Scattering,” Science 275, 1102 (1997). [CrossRef] [PubMed]

26.

A. Podlipensky, J. Lange, G. Seifert, H. Graener, and I. Cravetchi, “Second-harmonic generation from ellipsoidal silver nanoparticles embedded in silica glass,” Opt. Lett. 28, 716–718 (2003). [CrossRef] [PubMed]

27.

S.-P. Tai, Y. Wu, D.-B. Shieh, L.-J. Chen, K.-J. Lin, C.-H. Yu, S.-W. Chu, C.-H. Chang, X.-Y. Shi, Y.-C. Wen, K.-H. Lin, T.-M. Liu, and C.-K. Sun, “Molecular Imaging of Cancer Cells Using Plasmon-Resonant-Enhanced Third-Harmonic-Generation in Silver Nanoparticles,” Adv. Mater. 19, 4520–4523 (2007). [CrossRef]

28.

D. Yelin, D. Oron, S. Thiberge, E. Moses, and Y. Silberberg, “Multiphoton plasmon-resonance microscopy,” Opt. Express 11, 1385–1391 (2003), http://www.opticsinfobase.org/abstract.cfm?URI=oe-11-12-1385. [CrossRef] [PubMed]

29.

M. Lippitz, M. A. van Dijk, and M. Orrit, “Third-harmonic generation from single gold nanoparticles,” Nano Lett. 5, 799–802 (2005). [CrossRef] [PubMed]

30.

X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science 307, 538–544 (2005). [CrossRef] [PubMed]

31.

E. B. Voura, J. K. Jaiswal, H. Mattoussi, and S. M. Simon, “Tracking metastatic tumor cell extravasation with quantum dot nanocrystals and fluorescence emission-scanning microscopy,” Nat. Med. 10, 993–998 (2004). [CrossRef] [PubMed]

32.

A. K. Dharmadhikari, N. Kumbhojkar, J. A. Dharmadhikari, S. Mahamuni, and R. C. Aiyer, “Studies on third-harmonic generation in chemically grown ZnS quantum dots,” J. Phys.: Condens. Matter 11, 1363–1368 (1999). [CrossRef]

33.

D. Mohanta, G. A. Ahmed, A. Choudhury, F. Singh, D. K. Avasthi, G. Boyer, and G. A. Stanciu, “Scanning probe microscopy, luminescence and third harmonic generation studies of elongated CdS:Mn nanostructures developed by energetic oxygen-ion-impact,” Eur. Phys. J.: Appl. Phys. 35, 29–36 (2006). [CrossRef]

34.

S. Sauvage, P. Boucaud, F. Glotin, R. Prazeres, J. M. Ortega, A. Lemaitre, J. M. Gerard, and V. Thierry-Mieg, “Third-harmonic generation in InAs/GaAs self-assembled quantum dots,” Phys. Rev. B 59, 9830–9833 (1999). [CrossRef]

35.

C.-H. Yu, S.-P. Tai, C.-T. Kung, W.-J. Lee, Y.-F. Chan, H.-L. Liu, J.-Y. Lyu, and C.-K. Sun, “Molecular third-harmonic-generation microscopy through resonance enhancement with absorbing dye,” Opt. Lett. 33, 387–389 (2008). [CrossRef] [PubMed]

36.

M. A. Hines and P. Guyot-Sionnest, “Synthesis and characterization of strongly luminescing ZnS-Capped CdSe nanocrystals,” J. Phys. Chem. 100, 468–471 (1996). [CrossRef]

37.

C. B. Murray, D. J. Norris, and M. G. Bawendi, “Synthesis and characterization of nearly monodisperse CdE (E=sulfur, selenium, tellurium) semiconductor nanocrystallites,” J. Am. Chem. Soc. 115, 8706–8715 (1993). [CrossRef]

38.

C.-Y. Chen, S. R. Roffler, I.-S. Liu, C.-C. Liu, W.-F. Su, and F.-H. Changare preparing a manuscript to be called “Facile and efficient assembly of antibody-tagged quantum dots for membrane receptor targeting.”

39.

B. Dubertret, P. Skourides, D. J. Norris, V. Noireaux, A. H. Brivanlou, and A. Libchaber, “In Vivo Imaging of Quantum Dots Encapsulated in Phospholipid Micelles,” Science 298, 1759 (2002). [CrossRef] [PubMed]

40.

J. X. Cheng and X. S. Xie, “Green’s function formulation for third-harmonic generation microscopy,” J. Opt. Soc. Am. B: Opt. Phys. 19, 1604–1610 (2002). [CrossRef]

41.

M. J. Weber, Handbook of Optical Materials (CRC Press, 2003).

42.

J. M. Schins, T. Schrama, J. Squier, G. J. Brakenhoff, and M. Muller, “Determination of material properties by use of third-harmonic generation microscopy,” J. Opt. Soc. m. B: Opt. Phys. 19, 1627–1634 (2002). [CrossRef]

43.

J. T. Seo, S. M. Ma, Q. Yang, L. Creekmore, R. Battle, H. Brown, A. Jackson, T. Skyles, B. Tabibi, and W. Yu, “Large Resonant Third-order Optical Nonlinearity of CdSe Nanocrystal Quantum Dots,” J. Phys.: Conf. Ser. 38, 91–94 (2006). [CrossRef]

44.

D. Debarre, N. Olivier, and E. Beaurepaire, “Signal epidetection in third-harmonic generation microscopy of turbid media,” Opt. Express 15, 8913–8924 (2007), http://www.opticsinfobase.org/abstract.cfm?URI=oe-15-14-8913. [CrossRef] [PubMed]

45.

G. Veres, S. Matsumoto, Y. Nabekawa, and K. Midorikawa, “Enhancement of third-harmonic generation in absorbing media,” Appl. Phys. Lett. 81, 3714–3716 (2002). [CrossRef]

46.

C. J. Murphy, “Optical sensing with quantum dots,” Anal. Chem. 74, 520A–526A (2002). [CrossRef] [PubMed]

47.

M. Campiglio, A. Locatelli, C. Olgiati, N. Normanno, G. Somenzi, L. Vigano, M. Fumagalli, S. Menard, and L. Gianni, “Inhibition of proliferation and induction of apoptosis in breast cancer cells by the epidermal growth factor receptor(EGFR) tyrosine kinase inhibitor ZD 1839(‘Iressa’) is independent of EGFR expression level,” J. Cell. Physiol. 198, 259–268 (2004). [CrossRef]

48.

T. Faltus, J. Yuan, B. Zimmer, A. Kramer, S. Loibl, M. Kaufmann, and K. Strebhardt, “Silencing of the HER2/neu gene by siRNA inhibits proliferation and induces apoptosis in HER2/neu-overexpressing breast cancer cells,” Neoplasia 6, 786–795 (2004). [CrossRef]

49.

W. L. Xia, J. Bisi, J. Strum, L. H. Liu, K. Carrick, K. M. Graham, A. L. Treece, M. A. Hardwicke, M. Dush, Q. Y. Liao, R. E. Westlund, S. M. Zhao, S. Bacus, and N. L. Spector, “Regulation of survivin by ErbB2 signaling: Therapeutic implications for ErbB2-overexpressing breast cancers,” Cancer Res. 66, 1640–1647 (2006). [CrossRef] [PubMed]

50.

D. R. Emlet, R. Schwartz, K. A. Brown, A. A. Pollice, C. A. Smith, and S. E. Shackney, “HER2 expression as a potential marker for response to therapy targeted to the EGFR,” Br. J. Cancer 94, 1144–1153 (2006). [CrossRef] [PubMed]

51.

K. I. Pritchard, L. E. Shepherd, F. P. O’Malley, I. L. Andrulis, D. S. Tu, V. H. Bramwell, and M. N. Levine, “HER2 and responsiveness of breast cancer to adjuvant chemotherapy,” N. Engl. J. Med. 354, 2103–2111 (2006). [CrossRef] [PubMed]

52.

O. Thuerigen, A. Schneeweiss, G. Toedt, P. Warnat, M. Halm, H. Kramer, B. Brors, C. Rudlowski, A. Benner, F. Schuetz, B. Tews, R. Eils, H. P. Sinn, C. Sohn, and P. Lichter, “Gene expression signature predicting pathologic complete response with gemcitabine, epirubicin, and docetaxel in primary breast cancer,” J. Clin. Oncol. 24, 1839–1845 (2006). [CrossRef] [PubMed]

53.

G. L. Plosker and S. J. Keam, “Trastuzumab - A review of its use in the management of HER2-positive metastatic and early-stage breast cancer,” Drugs 66, 449–475 (2006). [CrossRef] [PubMed]

54.

S. S. Bacus, K. Kiguchi, D. Chin, C. R. King, and E. Huberman, “Differentiation of Cultured Human Breast Cancer Cells (AU-565 and MCF-7) Associated with Loss of Cell Surface HER-2/neu Antigen,” Mol. Carcinog. 3, 350–362 (1990). [CrossRef] [PubMed]

OCIS Codes
(180.5810) Microscopy : Scanning microscopy
(180.6900) Microscopy : Three-dimensional microscopy
(190.4160) Nonlinear optics : Multiharmonic generation

ToC Category:
Microscopy

History
Original Manuscript: February 22, 2008
Revised Manuscript: May 26, 2008
Manuscript Accepted: May 27, 2008
Published: June 13, 2008

Virtual Issues
Vol. 3, Iss. 7 Virtual Journal for Biomedical Optics

Citation
Chieh-Feng Chang, Chao-Yu Chen, Fu-Hsiung Chang, Shih-Peng Tai, Cheng-Ying Chen, Che-Hang Yu, Yi-Bing Tseng, Tsung-Han Tsai, I-Shuo Liu, Wei-Fang Su, and Chi-Kuang Sun, "Cell tracking and detection of molecular expression in live cells using lipid-enclosed CdSe quantum dots as contrast agents for epi-third harmonic generation microscopy," Opt. Express 16, 9534-9548 (2008)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-16-13-9534


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References

  1. D. Yelin and Y. Silberberg, "Laser scanning third-harmonic-generation microscopy in biology," Opt. Express 5, 169-175 (1999), http://www.opticsinfobase.org/abstract.cfm?URI=oe-5-8-169.
  2. S.-W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C.-Y. Lin, H.-J. Tsai, and C.-K. Sun, "In vivo developmental biology study using noninvasive multi-harmonic generation microscopy," Opt. Express 11, 3093-3099 (2003), http://www.opticsinfobase.org/abstract.cfm?URI=oe-11-23-3093.
  3. C.-K. Sun, S.-W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C.-Y. Lin, and H.-J. Tsai, "Higher harmonic generation microscopy for developmental biology," J. Struct. Biol. 147, 19-30 (2004). [CrossRef] [PubMed]
  4. D. Debarre, W. Supatto, E. Farge, B. Moulia, M. C. Schanne-Klein, and E. Beaurepaire, "Velocimetric third-harmonic generation microscopy: micrometer-scale quantification of morphogenetic movements in unstained embryos," Opt. Lett. 29, 2881-2883 (2004). [CrossRef]
  5. W. Supatto, D. Debarre, B. Moulia, E. Brouzes, J. L. Martin, E. Farge, and E. Beaurepaire, "In vivo modulation of morphogenetic movements in Drosophila embryos with femtosecond laser pulses," Proc. Nat. Acad. Sci. U.S.A. 102, 1047-1052 (2005). [CrossRef]
  6. D. Debarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, "Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy," Nat. Methods 3, 47-53 (2006). [CrossRef]
  7. T.-H. Tsai, C.-Y. Lin, H.-J. Tsai, S.-Y. Chen, S.-P. Tai, K.-H. Lin, and C.-K. Sun, "Biomolecular imaging based on far-red fluorescent protein with a high two-photon excitation action cross section," Opt. Lett. 31, 930-932 (2006). [CrossRef] [PubMed]
  8. S.-P. Tai, W.-J. Lee, D.-B. Shieh, P.-C. Wu, H.-Y. Huang, C.-H. Yu, and C.-K. Sun, "In vivo optical biopsy of hamster oral cavity with epi-third-harmonic-generation microscopy," Opt. Express 14, 6178-6187 (2006), http://www.opticsinfobase.org/abstract.cfm?URI=oe-14-13-6178. [CrossRef] [PubMed]
  9. S.-Y. Chen, C.-S. Hsieh, S.-W. Chu, C.-Y. Lin, C.-Y. Ko, Y.-C. Chen, H.-J. Tsai, C.-H. Hu, and C.-K. Sun, "Noninvasive harmonics optical microscopy for long-term observation of embryonic nervous system development in vivo," J. Biomed. Opt. 11, 054022 (2006). [CrossRef] [PubMed]
  10. C.-H. Yu, S.-P. Tai, C.-T. Kung, I.-J. Wang, H.-C. Yu, H.-J. Huang, W.-J. Lee, Y.-F. Chan, and C.-K. Sun, "In vivo and ex vivo imaging of intra-tissue elastic fibers using third-harmonic-generation microscopy," Opt. Express 15, 11167-11177 (2007), http://www.opticsinfobase.org/abstract.cfm?URI=oe-15-18-11167. [CrossRef] [PubMed]
  11. E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, "In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy," J. Microsc. 229, 141-150 (2008). [CrossRef] [PubMed]
  12. V. Barzda, C. Greenhalgh, J. Aus der Au, S. Elmore, J. van Beek, and J. Squier, "Visualization of mitochondria in cardiomyocytes by simultaneous harmonic generation and fluorescence microscopy," Opt. Express 13, 8263-8276 (2005), http://www.opticsinfobase.org/abstract.cfm?URI=oe-13-20-8263. [CrossRef] [PubMed]
  13. S.-W. Chu, I.-H. Chen, T.-M. Liu, C.-K. Sun, S.-P. Lee, B.-L. Lin, P.-C. Cheng, M.-X. Kuo, D.-J. Lin, and H.-L. Liu, "Nonlinear bio-photonic crystal effects revealed with multimodal nonlinear microscopy," J. Microsc. 208, 190-200 (2002). [CrossRef] [PubMed]
  14. C.-K. Sun, C.-C. Chen, S.-W. Chu, T.-H. Tsai, Y.-C. Chen, and B.-L. Lin, "Multiharmonic-generation biopsy of skin," Opt. Lett. 28, 2488-2490 (2003). [CrossRef] [PubMed]
  15. S.-P. Tai, T.-H. Tsai, W.-J. Lee, D.-B. Shieh, Y.-H. Liao, H.-Y. Huang, K. Zhang, H.-L. Liu, and C.-K. Sun, "Optical biopsy of fixed human skin with backward-collected optical harmonics signals," Opt. Express 13, 8231-8242 (2005), http://www.opticsinfobase.org/abstract.cfm?URI=oe-13-20-8231. [CrossRef] [PubMed]
  16. R. R. Anderson, and J. A. Parrish, "The Optics of Human Skin," J. Invest. Dermatol. 77, 13-19 (1981). [CrossRef] [PubMed]
  17. C.-K. Sun, Series in Advances in Biochemical Engineering/Biotechnology, Special Volume 95: Microscopy Techniques (Springer-Verlag, Berlin, 2005), Chap. "Higher harmonic generation microscopy."
  18. S.-W. Chu, S.-P. Tai, C.-L. Ho, C.-H. Lin, and C.-K. Sun, "High-resolution simultaneous three-photon fluorescence and third-harmonic-generation microscopy," Microsc. Res. Tech 66, 193-197 (2005). [CrossRef] [PubMed]
  19. G. O. Clay, A. C. Millard, C. B. Schaffer, J. Aus-der-Au, P. S. Tsai, J. A. Squier, and D. Kleinfeld, "Spectroscopy of third-harmonic generation: evidence for resonances in model compounds and ligated hemoglobin," J. Opt. Soc. Am. B: Opt. Phys. 23, 932-950 (2006). [CrossRef]
  20. R. D. Schaller, J. C. Johnson, and R. J. Saykally, "Nonlinear Chemical Imaging Microscopy: Near-Field Third Harmonic Generation Imaging of Human Red Blood Cells," Anal. Chem 72, 5361-5364 (2000). [CrossRef] [PubMed]
  21. H. Kano and S. Kawata, "Two-photon-excited fluorescence enhanced by a surface plasmon," Opt. Lett. 21, 1848-1850 (1996). [CrossRef] [PubMed]
  22. K. Kneipp, H. Kneipp, I. Itzkan, R. R. Dasari, and M. S. Feld, "Ultrasensitive chemical analysis by Raman spectroscopy," Chem. Rev. 99, 2957-2976 (1999). [CrossRef]
  23. B. Lamprecht, J. R. Krenn, A. Leitner, and F. R. Aussenegg, "Resonant and Off-Resonant Light-Driven Plasmons in Metal Nanoparticles Studied by Femtosecond-Resolution Third-Harmonic Generation," Phys. Rev. Lett. 83, 4421-4424 (1999). [CrossRef]
  24. T.-M. Liu, S.-P. Tai, C.-H. Yu, Y.-C. Wen, S.-W. Chu, L.-J. Chen, M. R. Prasad, K.-J. Lin, and C.-K. Sun, "Measuring plasmon-resonance enhanced third-harmonic ??(3) of Ag nanoparticles," Appl. Phys. Lett. 89, 043122 (2006). [CrossRef]
  25. S. Nie and S. R. Emory, "Probing Single Molecules and Single Nanoparticles by Surface-Enhanced Raman Scattering," Science 275, 1102 (1997). [CrossRef] [PubMed]
  26. A. Podlipensky, J. Lange, G. Seifert, H. Graener, and I. Cravetchi, "Second-harmonic generation from ellipsoidal silver nanoparticles embedded in silica glass," Opt. Lett. 28, 716-718 (2003). [CrossRef] [PubMed]
  27. S.-P. Tai, Y. Wu, D.-B. Shieh, L.-J. Chen, K.-J. Lin, C.-H. Yu, S.-W. Chu, C.-H. Chang, X.-Y. Shi, Y.-C. Wen, K.-H. Lin, T.-M. Liu, and C.-K. Sun, "Molecular Imaging of Cancer Cells Using Plasmon-Resonant-Enhanced Third-Harmonic-Generation in Silver Nanoparticles," Adv. Mater. 19, 4520-4523 (2007). [CrossRef]
  28. D. Yelin, D. Oron, S. Thiberge, E. Moses, and Y. Silberberg, "Multiphoton plasmon-resonance microscopy," Opt. Express 11, 1385-1391 (2003), http://www.opticsinfobase.org/abstract.cfm?URI=oe-11-12-1385. [CrossRef] [PubMed]
  29. M. Lippitz, M. A. van Dijk, and M. Orrit, "Third-harmonic generation from single gold nanoparticles," Nano Lett. 5, 799-802 (2005). [CrossRef] [PubMed]
  30. X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir, and S. Weiss, "Quantum dots for live cells, in vivo imaging, and diagnostics," Science 307, 538-544 (2005). [CrossRef] [PubMed]
  31. E. B. Voura, J. K. Jaiswal, H. Mattoussi, and S. M. Simon, "Tracking metastatic tumor cell extravasation with quantum dot nanocrystals and fluorescence emission-scanning microscopy," Nat. Med. 10, 993-998 (2004). [CrossRef] [PubMed]
  32. A. K. Dharmadhikari, N. Kumbhojkar, J. A. Dharmadhikari, S. Mahamuni, and R. C. Aiyer, "Studies on third-harmonic generation in chemically grown ZnS quantum dots," J. Phys.: Condens. Matter 11, 1363-1368 (1999). [CrossRef]
  33. D. Mohanta, G. A. Ahmed, A. Choudhury, F. Singh, D. K. Avasthi, G. Boyer, and G. A. Stanciu, "Scanning probe microscopy, luminescence and third harmonic generation studies of elongated CdS:Mn nanostructures developed by energetic oxygen-ion-impact," Eur. Phys. J.: Appl. Phys. 35, 29-36 (2006). [CrossRef]
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