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Optics Express

Optics Express

  • Editor: Michael Duncan
  • Vol. 14, Iss. 26 — Dec. 25, 2006
  • pp: 12930–12943

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Increased optical contrast in imaging of epidermal growth factor receptor using magnetically actuated hybrid gold/iron oxide nanoparticles

Jesse S. Aaron, Junghwan Oh, Timothy A. Larson, Sonia Kumar, Thomas E. Milner, and Konstantin V. Sokolov  »View Author Affiliations


Optics Express, Vol. 14, Issue 26, pp. 12930-12943 (2006)
http://dx.doi.org/10.1364/OE.14.012930


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Abstract

We describe a new approach for optical imaging that combines the advantages of molecularly targeted plasmonic nanoparticles and magnetic actuation. This combination is achieved through hybrid nanoparticles with an iron oxide core surrounded by a gold layer. The nanoparticles are targeted in-vitro to epidermal growth factor receptor, a common cancer biomarker. The gold portion resonantly scatters visible light giving a strong optical signal and the superparamagnetic core provides a means to externally modulate the optical signal. The combination of bright plasmon resonance scattering and magnetic actuation produces a dramatic increase in contrast in optical imaging of cells labeled with hybrid gold/iron oxide nanoparticles.

© 2006 Optical Society of America

1. Introduction

Exogenous contrast agents are widely used to increase signal intensity and specificity during optical interrogation of biological materials. Organic fluorescent dyes are traditional contrast enhancing molecules for in vitro and in vivo optical imaging [1–4

B. N. G. Giepmans, S. R. Adams, M. H. Ellisman, and R. Y. Tsien, “The Fluorescent Toolbox for Assessing Protein Location and Function,” Science (Washington, DC, United States) 312, 217–224 (2006). [CrossRef] [PubMed]

]. Recent advances in nanotechnology have led to the development of novel bright contrast agents, including quantum dots [5–9

X. Gao, Y. Cui, R. M. Levenson, L. W. K. Chung, and S. Nie, “In vivo cancer targeting and imaging with semiconductor quantum dots,” Nature Biotechnology 22, 969–976 (2004). [CrossRef] [PubMed]

] and plasmonic nanoparticles [10–15

J. Yguerabide and E. E. Yguerabide, “Resonance light scattering particles as ultrasensitive labels for detection of analytes in a wide range of applications,” Journal of Cellular Biochemistry, 71–81 (2001).

]. Progress in nanomaterial chemistry has allowed synthesis of semiconductor quantum dots with increased fluorescence efficiencies [16

S. V. Kershaw, M. Burt, M. Harrison, A. Rogach, H. Weller, and A. Eychmuller, “Colloidal CdTe/HgTe quantum dots with high photoluminescence quantum efficiency at room temperature,” Appl. Phys. Lett. 75, 1694–1696 (1999). [CrossRef]

], tunable emission bands [17

M. Han, X. Gao, J. Z. Su, and S. Nie, “Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules,” Nature Biotechnology 19, 631–635 (2001). [CrossRef] [PubMed]

], and relatively slow photobleaching rates [6

W. C. Chan and S. Nie, “Quantum dot bioconjugates for ultrasensitive nonisotopic detection,” Science 281, 2016–2018. (1998). [CrossRef] [PubMed]

]. On the other hand, it was demonstrated that plasmonic metal nanoparticles offer additional advantages over luminescent quantum dots including significantly larger optical cross sections, complete resistance to photobleaching, and non-toxic constituent materials [10–15

J. Yguerabide and E. E. Yguerabide, “Resonance light scattering particles as ultrasensitive labels for detection of analytes in a wide range of applications,” Journal of Cellular Biochemistry, 71–81 (2001).

, 18

P. Alivisatos, “The use of nanocrystals in biological detection,” Nature Biotechnology 22, 47–52 (2004). [CrossRef] [PubMed]

].

Strategies using antibody or aptamer targeting molecules provide molecular specificity to optical imaging [19

C. F. Meares, A. J. Chmura, M. S. Orton, T. M. Corneillie, and P. A. Whetstone, “Molecular tools for targeted imaging and therapy of cancer,” Journal of Molecular Recognition 16, 255–259 (2003). [CrossRef] [PubMed]

, 20

R. Pasqualini and E. Ruoslahti, “Organ targeting in vivo using phage display peptide libraries,” Nature (London) 380, 364–366 (1996). [CrossRef] [PubMed]

]. However, most biological systems are very complex both in composition and morphology. Thus, endogenous scatterers and fluorophors can account for a significant portion of the total signal intensity causing problems in molecular specific optical imaging, especially in vivo [5

X. Gao, Y. Cui, R. M. Levenson, L. W. K. Chung, and S. Nie, “In vivo cancer targeting and imaging with semiconductor quantum dots,” Nature Biotechnology 22, 969–976 (2004). [CrossRef] [PubMed]

]. Molecular specific contrast agents would be better utilized if novel optical methods were employed that concurrently reduce or prevent unwanted background signal. It was demonstrated that the relative contribution of the endogenous background signal can be reduced using the effect of plasmon resonance coupling between gold nanoparticles [12

K. Sokolov, M. Follen, J. Aaron, I. Pavlova, A. Malpica, R. Lotan, and R. Richards-Kortum, “Real-time vital optical imaging of precancer using anti-epidermal growth factor receptor antibodies conjugated to gold nanoparticles,” Cancer Research 63, 1999–2004 (2003). [PubMed]

, 15

K. Sokolov, J. Aaron, B. Hsu, D. Nida, A. Gillenwater, M. Follen, C. MacAulay, K. Adler-Storthz, B. Korgel, M. Descour, R. Pasqualini, W. Arap, W. Lam, and R. Richards-Kortum, “Optical systems for in vivo molecular imaging of cancer,” Technology in Cancer Research & Treatment 2, 491–504 (2003). [PubMed]

]. The dipole-dipole coupling between closely spaced gold particles produces a red wavelength shift in their scattering and extinction cross sections [21

P. K. Aravind, A. Nitzan, and H. Metiu, “The interaction between electromagnetic resonances and its role in spectroscopic studies of molecules adsorbed on colloidal particles or metal spheres,” Surface Science 110, 189–204 (1981). [CrossRef]

, 22

W. Rechberger, A. Hohenau, A. Leitner, J. R. Krenn, B. Lamprecht, and F. R. Aussenegg, “Optical properties of two interacting gold nanoparticles,” Opt. Commun. 220, 137–141 (2003). [CrossRef]

]. This property was exploited for development of ultrasensitive DNA assays in vitro [23–25

R. Elghanian, J. J. Storhoff, R. C. Mucic, R. L. Letsinger, and C. A. Mirkin, “Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles,” Science 277, 1078–1080 (1997). [CrossRef] [PubMed]

]. Recently, we used 12 nm gold nanoparticles conjugated to monoclonal antibodies specific for epidermal growth factor receptor (EGFR) to label living cancer cells [12

K. Sokolov, M. Follen, J. Aaron, I. Pavlova, A. Malpica, R. Lotan, and R. Richards-Kortum, “Real-time vital optical imaging of precancer using anti-epidermal growth factor receptor antibodies conjugated to gold nanoparticles,” Cancer Research 63, 1999–2004 (2003). [PubMed]

]. Although isolated nanoparticles have a maximum scattering cross section in the green optical region, labeled cells exhibit a very strong scattering signal in the red. This red-shift behavior was attributed to the dipole-dipole coupling of gold bioconjugates bound to EGFR molecules on the cell surface [12

K. Sokolov, M. Follen, J. Aaron, I. Pavlova, A. Malpica, R. Lotan, and R. Richards-Kortum, “Real-time vital optical imaging of precancer using anti-epidermal growth factor receptor antibodies conjugated to gold nanoparticles,” Cancer Research 63, 1999–2004 (2003). [PubMed]

]. The effect of plasmon resonance coupling affords an opportunity to spectrally reject the background associated with endogenous cellular scattering which is predominant in the blue spectral region.

Another effective strategy to reduce unwanted background signal has been demonstrated by Oldenburg, et al. [26

L. Oldenburg Amy, R. Gunther Jillian, and A. Boppart Stephen, “Imaging magnetically labeled cells with magnetomotive optical coherence tomography,” Opt. Lett. 30, 747–749. (2005). [CrossRef]

] in optical coherence tomography (OCT) imaging of macrophages loaded with magnetic particles. The approach is based on inducing a magnetically actuated movement in cells containing magnetic iron oxide microparticles. Subsequent implementation of a simple frame subtraction algorithm reduces the background signal associated with the surrounding non-magnetic media. A similar approach was used by Anker et al. [27

J. N. Anker and R. Kopelman, “Magnetically modulated optical nanoprobes,” Appl. Phys. Lett. 82, 1102–1104 (2003). [CrossRef]

]. In this scheme, polystyrene microbeads loaded with a dye and a ferromagnetic material were sputter-coated with aluminum or gold leaving a small uncoated area on the surface of the beads. Then, the microbeads were used for optical imaging and exhibited a bright fluorescence signal only when they were magnetically oriented with a metal coated side facing away from a detector. The metal coating blocked the fluorescent emission when the metal-coated side was oriented towards the detector, and image subtraction removed non-specific signals. In another study, superparamagnetic iron oxide nanoparticles (SPIO) have been magnetically actuated in mouse liver tissue and detected using ultrasound imaging [28

J. Oh, M. Feldman, J. Kim, C. Condit, S. Emelianov, and T. E. Milner, “Detection of magnetic nanoparticles in tissue using magneto-motive ultrasound” Nanotechnology 17, 8 (2006). [CrossRef]

].

Recently, a new type of nanomaterial - magnetic/gold composite nanoparticles have been synthesized by several different routes [29–31

I. Stoeva Savka, F. Huo, J.-S. Lee, and A. Mirkin Chad, “Three-layer composite magnetic nanoparticle probes for DNA,” Journal of the American Chemical Society 127, 15362–15363. (2005). [CrossRef]

]. Lin et al. [31

J. Lin, W. Zhou, A. Kumbhar, J. Wiemann, J. Fang, E. E. Carpenter, and C. J. O’Connor, “Gold-Coated Iron (Fe@Au) Nanoparticles: Synthesis, Characterization, and Magnetic Field-Induced Self-Assembly,” Journal of Solid State Chemistry 159, 26–31 (2001). [CrossRef]

] utilized an inverse micelle technique to form crystalline iron cores within micelles and subsequently adding chlorauric acid and sodium borohydride to obtain gold-coated iron. The inverse micelle technique creates particles with a narrow size distribution, but further steps must be taken to transfer them to aqueous solution for subsequent functionalization. The magnetic properties of these particles were further characterized in [32

S.-J. Cho, B. R. Jarrett, A. Y. Louie, and S. M. Kauzlarich, “Gold-coated iron nanoparticles: a novel magnetic resonance agent for T1 and T2 weighted imaging,” Nanotechnology 17, 640–644 (2006). [CrossRef]

]. It is important to note that the gold coating does not prevent the iron core from oxidizing over a period of several weeks [33

S.-J. Cho, A. M. Shahin, G. J. Long, J. E. Davies, K. Liu, F. Grandjean, and S. M. Kauzlarich, “Magnetic and Moessbauer Spectral Study of Core/Shell Structured Fe/Au Nanoparticles,” Chemistry of Materials 18, 960–967 (2006). [CrossRef]

]. Stoeva et al. [34

S. I. Stoeva, F. Huo, J.-S. Lee, and C. A. Mirkin, “Three-Layer Composite Magnetic Nanoparticle Probes for DNA,” Journal of the American Chemical Society 127, 15362–15363 (2005). [CrossRef] [PubMed]

] synthesized ~200 nm diameter three-layer nanoparticles with a Fe2O3 layer between a SiO2 core and an outer gold shell in a two phase process. Once in aqueous phase, these particles were functionalized with oligonucleotides and shown to reversibly bind via DNA hybridization. Wang et al. [35

H. Wang, D. W. Brandl, F. Le, P. Nordlander, and N. J. Halas, “Nanorice: A Hybrid Plasmonic Nanostructure,” Nano Letters 6, 827–832 (2006). [CrossRef] [PubMed]

] followed a similar synthesis route to produce iron oxide/gold core/shell nanorods with a final length of 250 nm. The synthesis developed by Lyon et al. [30

J. L. Lyon, D. A. Fleming, M. B. Stone, P. Schiffer, and M. E. Williams, “Synthesis of Fe Oxide Core/Au Shell Nanoparticles by Iterative Hydroxylamine Seeding,” Nano Letters 4, 719–723 (2004). [CrossRef]

] is entirely aqueous and was adapted from an iterative hydroxylamine seeding technique originally suggested in [36

K. R. Brown and M. J. Natan, “Hydroxylamine Seeding of Colloidal Au Nanoparticles in Solution and on Surfaces,” Langmuir 14, 726–728 (1998). [CrossRef]

]. Jeong et al. [37

J. Jeong, T. H. Ha, and B. H. Chung, “Enhanced reusability of hexa-arginine-tagged esterase immobilized on gold-coated magnetic nanoparticles,” Analytica Chimica Acta 569, 203–209 (2006). [CrossRef]

] used the hydroxyl amine seeding protocol to produce a magnetic colloid suspension and then attached a functional enzyme to the nanoparticles to magnetically decant and recover the enzyme for reuse. Wang et al. [38

F. H. Wang, I. H. Lee, N. Holmstroem, T. Yoshitake, D. K. Kim, M. Muhammed, J. Frisen, L. Olson, C. Spenger, and J. Kehr, “Magnetic resonance tracking of nanoparticle labelled neural stem cells in a rat’s spinal cord,” Nanotechnology 17, 1911–1915 (2006). [CrossRef]

] used an inverse micelle technique to synthesize magnetic gold particles that could be loaded into rat embryonic neural cells for in-vivo MRI imaging.

Here, we describe a new approach for molecular specific optical imaging in-vitro that combines the advantages of molecularly targeted plasmonic nanoparticles and magnetic actuation. This combination is achieved through hybrid nanoparticles with a superparamagnetic core surrounded by a gold layer. The nanoparticles were conjugated with monoclonal antibodies for molecular recognition. The hybrid nature of these particles provides new opportunities for optical contrast enhancement. The addition of the gold layer leads to three important advantages: (1) strong optical signal that facilitates detection and digital processing; (2) tunable optical resonances; and (3) a convenient surface for conjugation of probe molecules [39

W. D. Geoghegan and G. A. Ackerman, “Adsorption of horseradish peroxidase, ovomucoid and antiimmunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ agglutinin and goat antihuman immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and application,” Journal of Histochemistry and Cytochemistry 25, 1187–1200 (1977). [CrossRef] [PubMed]

, 40

L. Liu and H. Elwing, “Complement activation on thiol-modified gold surfaces,” Journal of Biomedical Materials Research 30, 535–541 (1996). [CrossRef] [PubMed]

]. The iron oxide core provides a magnetically susceptible component which can be exploited to periodically actuate the magnetic particles attached to cells in the field of view and, therefore, allows use of an external magnetic field for modulation of the optical signal. We demonstrate that opto-magnetic hybrid nanoparticles can be used to increase optical contrast in cancer cell imaging. This report is focused on EGFR - one of the hallmarks of carcinogenesis. EGFR has been found to be over-expressed in many types of cancers including lung, breast, bladder, cervix, and oral cavity [41

D. Hanahan and R. A. Weinberg, “The hallmarks of cancer,” Cell (Cambridge, Massachusetts) 100, 57–70 (2000). [CrossRef] [PubMed]

].

2. Methods

2.1 Iron oxide/gold hybrid nanoparticles

Magnetically susceptible plasmonic nanoparticles were synthesized using the method described in [30

J. L. Lyon, D. A. Fleming, M. B. Stone, P. Schiffer, and M. E. Williams, “Synthesis of Fe Oxide Core/Au Shell Nanoparticles by Iterative Hydroxylamine Seeding,” Nano Letters 4, 719–723 (2004). [CrossRef]

]. Briefly, 9 nm magnetite (Fe3O4) particles were formed via co-reduction of FeCl2 and FeCl3 in an aqueous NaOH solution. The Fe3O4 cores were oxidized to primarily Fe2O3 by boiling in a 0.01M HNO3 solution. X-ray diffraction measurements (not shown) of the prepared magnetic cores were characteristic for maghemite, or γ-Fe2O3. Subsequently, a ca. 20nm thick gold shell was deposited using the hydroxylamine seeding method [36

K. R. Brown and M. J. Natan, “Hydroxylamine Seeding of Colloidal Au Nanoparticles in Solution and on Surfaces,” Langmuir 14, 726–728 (1998). [CrossRef]

]. This procedure involves sequential additions of HAuCl3 in the presence of citrate and hydroxylamine. It was shown that hydroxylamine confines the reduction of Au3+ ions to the pre-existing surface of iron oxide particles, thereby largely preventing the nucleation of pure gold particles in solution. The iron seeds and hybrid iron oxide/gold nanoparticles were characterized using a Philips EM 208 Transmission Electron Microscope (TEM) equipped with an AMT Advantage HR 1MB digital camera detector. Fig. 1(A) shows transmission electron micrographs of the Fe2O3 core nanoparticles before the addition of gold and Fig. 1(B) reveals the morphology after deposition of the gold layer. The addition of gold results in an approximately 5-fold increase in particle diameter. The average diameter of the iron oxide/gold nanoparticles was 50 nm with a standard deviation of 14 nm. The cause for the relatively large size distribution of the resulting nanoparticles is not very well understood [28

J. Oh, M. Feldman, J. Kim, C. Condit, S. Emelianov, and T. E. Milner, “Detection of magnetic nanoparticles in tissue using magneto-motive ultrasound” Nanotechnology 17, 8 (2006). [CrossRef]

] and it presents a technical challenge that remains to be fully addressed. Possible aggregation of the iron oxide core particles before gold deposition might be part of the problem. The process of gold deposition was also monitored using an UV-Vis spectrophotometer (BioTek Synergy HT micro-titer plate spectrometer), as shown in Fig. 1(C). Before the addition of the gold shell, the extinction properties of the superparamagnetic particles are consistent with sub-wavelength sized dielectric spheres. However, upon addition of the gold layer onto the iron oxide core, the extinction spectrum changes markedly, displaying a plasmon resonance peak at 540nm.

We ran theoretical simulations to model the scattering and absorption properties of the gold/iron oxide nanoparticles. Core-shell composite particle simulations were implemented using custom Matlab/C++ codes based on the equations from [42

A. L. Aden and M. Kerker, “Scattering of Electromagnetic Waves from Two Concentric Spheres,” J. Appl. Phys. 22, 1242–1246 (1951). [CrossRef]

]. Dielectric functions used are based on the experimental data from [43

P. B. Johnson and R. W. Christy, “Optical constants of the noble metals,” Physical Review B: Solid State 6, 4370–4379 (1972). [CrossRef]

], with corrections for the effect of particle size as detailed in [44

U. Kreibig, “Properties of Small Particles in Insulating Matrices,” in Contribution of Clusters Physics to Material Science and Technology From Isolated Clusters to Aggregated Materials, J. Davenas and P. M. Rabette, eds. (Klewer Academic Publishers, New York, NY, 1986), pp. 373–423.

]. Results from these codes have been extensively compared to composite particle simulations in the literature. In order to simulate the effects of a statistical distribution of particle sizes, the output from the cross section codes was further integrated using a Gauss-Lobatto adaptive quadrature algorithm. The simulated extinction spectrum is in excellent agreement with our measurements (Fig 1 C and D). The simulations showed that the peak absorption, extinction, and scattering wavelengths are 534nm, 537nm, and 550nm, respectively. The total scattering from the hybrid nanoparticles represents about 20% of the total integrated extinction.

2.2 Antibody conjugation

The hybrid nanoparticles were conjugated to anti-EGFR monoclonal antibodies (clone 29.1.1, Sigma) for molecular specific imaging. Antibodies were attached to gold nanoparticles via a conjugation linker that consists of a short polyethylene glycol (PEG) chain terminated at one end by a hydrazide moiety, and at the other end by two thiol groups. First, antibodies at a concentration of 1mg/mL were exposed to 10mM NaIO4 in a 40mM HEPES pH 7.4 solution for 30–40 minutes at room temperature, thereby oxidizing the hydroxyl moieties on the antibodies’ Fc region to aldehyde groups. The formation of the aldehyde groups was colorimetrically confirmed using a standard assay with an alkaline Purpald solution (Sigma). Then, excess hydrazide-PEG-thiol linker was added to the oxidized antibodies and allowed to react for 20 minutes. The hydrazide portion of the PEG linker interacts with aldehyde groups on the antibodies to form a stable linkage. In this procedure a potential loss of antibody function is avoided because the linker can not interact with the antibody’s target-binding region, which contains no glycosylation. The unreacted linker was removed by filtration through a 100,000 MWCO filter (Millipore). After purification, the modified antibodies were mixed with gold nanoparticles in 40mM HEPES (pH 7.4) for 20 minutes at room temperature. During this step a stable bond is formed between the gold surface and the linker’s thiol groups. Afterward, monofunctional PEG-thiol molecules were added to passivate the remaining nanoparticle surface. Finally, the conjugates were centrifuged at 2800 rcf for 45 minutes and resuspended in 1× PBS.

Fig. 1. TEM images of 9nm Fe2O3 nanoparticles before (A) and after (B) deposition of the metallic gold layer. Images were collected at 80keV acceleration and 180,000× direct magnification. Scale bars are 200nm. In (C), UV-Vis extinction spectra are shown of a suspension of 9nm Fe2O3 nanoparticles (blue) and the same suspension after deposition of ca. 20nm gold layer (pink). Bare Fe2O3 particles show typical inverse-power law type extinction properties. The addition of gold to the surface results in the appearance of a characteristic plasmon resonance peak at ca. 540nm. In (D), theoretical simulations show the relative contribution of absorption (pink) and scattering (blue) to the total extinction (black) of the nanoparticles. The calculated maximum wavelengths for absorption, extinction, and scattering are 534nm, 537nm, and 550nm, respectively and include the effect of particle size distribution as determined by TEM.

2.3 Cell culture model

EGFR over-expressing A-431 cells [45

D. S. Lidke, P. Nagy, R. Heintzmann, D. J. Arndt-Jovin, J. N. Post, H. E. Grecco, E. A. Jares-Erijman, and T. M. Jovin, “Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction,” Nature Biotechnology 22, 198–203 (2004). [CrossRef] [PubMed]

] were used to demonstrate molecular specific imaging with hybrid iron oxide/gold nanoparticles. Cells were cultured in DMEM plus 10% FBS at 37°C in a 5% CO2 environment. For labeling experiments, the cells were suspended in phenol-free DMEM, mixed with the nanoparticle-antibody conjugates, and allowed to react for 20–30 minutes under mild agitation at room temperature. Typically, 200–300µL of a cell suspension (~105 cells/mL) were mixed with an equal volume of nanoparticles suspended at approximately 1010 particles/mL. The labeled cells were washed in phenol-free DMEM and resuspended in an isotonic 1% gelatin solution. The gelatin provides a viscous environment that is more similar to in vivo conditions than pure tissue culture media and also prevents cells from electrostatically adhering to the glass coverslip during imaging. In addition to cells labeled with hybrid nanoparticles we included two internal negative controls: unlabeled A-431 cells and cells labeled with 40nm pure gold nanoparticles. Because of relatively small optical property differences between 40 nm pure gold and 50 nm magnetic gold nanoparticles (only ca. 10 nm separation in extinction spectra maxima) we conjugated pure gold particles with fluorescently labeled anti-EGFR monoclonal antibodies. Therefore, pure-gold particles exhibited a strong fluorescence signal, while the gold/iron oxide particles did not, allowing easy discrimination between the two populations of labeled cells. We used AlexaFluor 488 as the fluorescent tag and a standard labeling kit available from Molecular Probes to fluorescently label antibodies. The controls were prepared in the same manner as cells labeled with hybrid nanoparticles and all three cell types were mixed together in 1:1:1 ratio. An aliquot of this mixture was placed on a microscope slide for optical measurements.

2.4 Imaging system

Samples were imaged using a Leica DM 6000 upright microscope in epi-illuminated darkfield mode. A 75W Xenon light source was used for illumination. Images were collected through a 20× darkfield/brightfield objective with a 0.5 collection NA, and detected using a Q-Imaging Retiga EXi ultra-sensitive 12-bit CCD camera. Time-course images of magnetically actuated cells were taken in monochrome mode at approximately 10 frames per second. Hyperspectral imaging was used to measure the spectral differences between labeled and unlabeled cells. The hyperspectral imaging system (PARISS, LightForm, Inc.) incorporates a slit and a prism dispersion configuration. In this scheme, the sample is laterally scanned using a piezoelectric stage, with the slit allowing a ~1µm wide portion of the image through the imaging system. Each line of the image is spectrally dispersed via the prism and projected onto a two dimensional CCD detector. The device allows for a spectral range of approximately 350–850nm, and 1 nm spectral resolution. A microscopically clean aluminum mirror was used to collect the spectral profile of the light source, which was used to normalize the spectra recorded from cells. Fluorescence imaging was performed in epi-mode using a 490nm excitation/510nm emission fluorescence filter cube (Chroma).

2.5 Statistical Image Analysis

For each type of cell (magnetically labeled, pure gold labeled, and unlabeled), as well as for each illumination condition (white light and 635nm band-pass illumination) and for magnetically actuated and un-actuated, 10 cells or more were analyzed. To calculate average signal intensities each cell was manually segmented from the image, the signal background subtracted, and the average non-zero pixel intensity values were calculated. Then an average signal and standard deviation were determined for each cell type and illumination condition. A one-tailed, paired t-test (assuming unequal variances) was performed among the three cell populations. Then, the resulting T statistic then was used to calculate a p-value. Calculations were repeated in both Matlab and Excel for confirmation.

2.6 Magnetic actuation

A solenoid electromagnet (Ledex 6EC) with a cone shaped ferrite core was used to magnetically actuate the samples. The electromagnet was driven by a power supply and current amplifier, which delivered up to 960W to the coil. The field strength at the tip of the magnet was 0.7 T and the field gradient in z-direction from the tip of the core extending 1 mm outward was 220 T/m. The electromagnet was attached to a motorized translation stage (Aerotech) and driven by a programmable controller that permitted sinusoidal movement with a user determined frequency and amplitude. The motion amplitude was adjusted to approximately one full field of view. Care was taken to ensure that the moving stage was mechanically-isolated from the microscope and its vibration isolation table. Any sample movements due to vibrations caused by the moving stage were minimized. Figure 2 outlines the experimental setup.

Fig. 2. Experimental setup. Cells were mounted on a microscope slide and imaged in reflected darkfield mode using a Leica DM 6000 upright microscope. A solenoid electromagnet with a cone-shaped ferrite core was attached to a programmable piezoelectric translation stage, and placed beneath the sample stage. Translation stage motion oscillated in a sinusoidal fashion with a user-definable amplitude and frequency. The magnet was powered by a power supply and amplifier delivering up to 960 W. The solenoid and motorized translation stage assembly was mechanically isolated from the microscope, which sat on a vibration isolation table.

3. Results

Figure 3(A) shows a color dark-field reflectance image of the A-431 cell mixture which consists of unlabeled cells (blue arrows), cells labeled with 50nm gold/iron oxide nanoparticles (red arrows) and with 40nm pure gold nanoparticles (green arrows). Labeled cell types were differentiated from one another using a fluorescent tag (AlexaFluor 488, Molecular Probes) that was attached to the monoclonal antibodies conjugated with 40nm pure gold nanoparticles and was absent on the hybrid nanoparticles. The unlabeled cells appear blue due to the characteristic intrinsic cellular scattering. The labeled cells exhibit dim green regions and bright easily identifiable regions with different shades of orange. The green tinge is the color of the isolated nanoparticles and corresponds to regions with low density of the contrast agents. The orange color corresponds to the closely spaced assemblies of anti-EGFR gold conjugates which interact with EGFR receptors on the cytoplasmic membrane of A-431 cells. The intensity difference between the labeled and unlabeled cells which is achieved under white light illumination can be additionally improved if a 635±15nm band-pass filter is placed into the illumination path, as shown in Fig. 3(B). This is possible because the endogenous scattering of cells in Fig. 3(C), blue line, is significantly reduced in the red optical region [46

V. Backman, V. Gopal, M. Kalashnikov, K. Badizadegan, R. Gurjar, A. Wax, I. Georgakoudi, M. Mueller, C. W. Boone, R. R. Dasari, and M. S. Feld, “Measuring cellular structure at submicrometer scale with light scattering spectroscopy,” IEEE J. Sel. Top. Quantum Electron. 7, 887–893 (2001). [CrossRef]

]. In addition, cells labeled with 50 nm hybrid particles display a prominent scattering peak in the red region at approximately 690 nm, shown in Fig. 3(C), red line. Similar behavior is observed with cells labeled with 40nm pure gold nanoparticles seen in Fig. 3(C), green line.

Fig. 3. Darkfield images of a 1:1:1 mixture of A-431 cells labeled with 40nm anti-EGFR gold nanoparticles (indicated by green arrows), 50nm anti-EGFR gold/iron oxide nanoparticles (indicated by red arrows), and unlabeled cells (indicated by blue arrows) obtained using: (A) white light illumination; and (B) a 630±15nm bandpass filter. Images were acquired with a 20x darkfield/brightfield objective with a 0.5 collection NA. (C) Scattering spectra of cells labeled with 50 nm hybrid nanoparticles (red line), 40nm pure gold nanoparticles (green line) and of unlabeled cells (blue line). A fluorescent tag (AlexaFluor 488, Molecular Probes) was attached to the pure gold-antibody conjugates in order to differentiate between the two types of labeled cells.

Despite the unique contrast-enhancing mechanism afforded by the plasmon resonance coupling of gold nanoparticles, unlabeled cells can still be detected in images obtained using both white light and red band-pass illumination, as shown in Fig. 3(A) and (B). To further increase contrast between labeled and unlabeled cells, we explored the magnetic component of the hybrid contrast agents. We demonstrate that cells labeled with magnetic gold nanoparticles can be easily discriminated from both the unlabeled cells and cells labeled with pure gold particles. The experimental set-up for magnetic actuation of labeled cells (Fig. 2) is based on the following principles. The force exerted on the nanoparticles is proportional to the gradient of the square of the magnetic field magnitude [26

L. Oldenburg Amy, R. Gunther Jillian, and A. Boppart Stephen, “Imaging magnetically labeled cells with magnetomotive optical coherence tomography,” Opt. Lett. 30, 747–749. (2005). [CrossRef]

], and acts in the direction of increasing gradient; thus the iron oxide nanoparticles tend to move towards the ferrite tip in the solenoid when current is applied. By oscillating the tip of the solenoid in a sinusoidal fashion in the horizontal or x-direction, the changing direction of force exerted on the nanoparticle-labeled cells causes an oscillating displacement in the cells’ position. The magnitude of the oscillation was approximately 1 field of view, or 500 microns. Magnetically induced movement of cells labeled with iron-oxide/gold nanoparticles is shown in Fig. 4.

Fig. 4. AVI file showing the magnetically induced movement of gold-iron oxide nanoparticle labeled A-431 cells captured under 20× magnification. Note no movement of unlabeled cells (which appear dim) and pure-gold labeled cells (identified by the overlaid green fluorescence signal); the magnetically labeled cell clearly responds to the oscillating magnetic field by fluctuating laterally in the horizontal direction, and also by translating in the vertical direction toward the ferrite tip which is located outside and above the field of view shown in this figure. The cell also shows some “rocking” movement which is a result of variations in the magnetic force in z-direction with movement of the solenoid. Because the cell has unevenly distributed magnetic gold nanoparticles it creates a net torque that forces cell to “rock”. Images were captured and replayed at 10 frames per second. The movie shows approximately 4 oscillations of the solenoid. [Media 1]

Images were collected and replayed at 10 frames per second. As can be seen, the horizontal translation of the solenoid tip causes horizontal fluctuations in the cell position due to interaction between the electromagnet and the magnetic nanoparticles which are attached to EGFR molecules on cellular surface. In addition, however, there also exists a y-component of the magnetic force due to the fact that the ferrite tip is not positioned directly beneath the cell labeled with hybrid nanoparticles. Cells that are not perfectly aligned in z-direction with the solenoid tip experience a non-oscillatory component in the y-direction. It is also important to note that the solenoid exerts the bulk of its force in the z-direction, parallel to the microscope’s optical axis. While cell translation is confined in this direction due to the presence of the microscope slide and coverslip, it may produce an overall torque on cells that have an uneven distribution on particles on their surfaces. This torque will result in signal fluctuations at the same frequency as the solenoid oscillation, and thus will contribute to the overall magnetic actuation effect. Any significant signal fluctuations at the modulation frequency of the solenoid are absent in the case of unlabeled cells and cells labeled with pure gold nanoparticles or background.

To analyze the specific frequency components of the acquired signals, a fast Fourier transform (FFT) was performed at each pixel of the acquired images in the time domain and power spectra were calculated at each pixel position. Oscillation frequencies of the magnet and the total number of acquired images were chosen to avoid any aliasing effects. The precise sampling frequency was calculated via a time stamp generated in each image file that is accurate to 0.001 seconds. Figures 5(A) and (B) show monochrome darkfield images of a mixture of cells that are labeled with gold/iron oxide particles, with pure gold particles, as well as unlabeled cells. The samples were subjected to a magnetic field oscillation with frequencies of 0.9Hz (sample in A) and 1.9Hz (sample in B). After data acquisition, images were analyzed in Matlab. Figures 5(C) and (D) show examples of frequency spectra from magnetic gold labeled (red line), pure gold labeled (green line), and unlabeled cells (blue line), for magnetic oscillations with frequencies 0.9 and 1.9 Hz, respectively. Frequency spectra of signals recorded from cells labeled with the magnetic/gold nanoparticles display a prominent peak at the corresponding stage oscillation frequency. Such a peak is not apparent in the case of unlabeled cells or cells labeled with pure gold nanoparticles, indicating that these cells are not displaced by the spatiotemporally oscillating magnetic field.

These results also suggest that secondary effects such as localized temperature-induced convection currents within the gelatin matrix are minimal. Time-varying signal intensities are predominant in only those regions of interest which contain the magnetically labeled cells. To isolate magnetically modulated components in the acquired images, we used a Hanning window method implemented in Fourier space [47

F. J. Harris, “On the Use of Windows for Harmonic Analysis with the Discrete Fourier Transform,” Proceedings of the IEEE 66, 33 (1978). [CrossRef]

]. First, image series were subjected to the appropriate window function and, then, to an inverse Fourier transform at each pixel in the time-dimension. Finally, images were rescaled via a simple linear multiplier to maximize the pixel intensity range. Presented images were not subjected to any thresholding procedure, which would artificially distort image contrast. Figures 5(E) and (F) show the same fields of view as Fig. 5 (A) and (B), respectively, after digital filtering at the appropriate frequencies (0.9Hz and 1.9Hz) and rescaling. As a result of this treatment, signals associated with unlabeled cells, and pure gold-labeled cells are no longer apparent in images filtered at both 0.9Hz and 1.9Hz, as shown in Fig. 5(E) and (F), respectively.

Implementation of frequency domain filtering results in greater contrast enhancement as compared to purely optical methods. To demonstrate this contrast enhancement, Figure 6(A) shows pixel intensity profiles that were drawn across images of magnetic gold-labeled (red line), pure-gold labeled (green line) and unlabeled cells (blue line) which were obtained under different illumination conditions and with the combination of the 635 nm illumination and frequency domain filtering. We also calculated the average signal intensities for each of the three cell populations in images that were acquired under four different acquisition conditions: (1) white light illumination, (2) 635/15nm bandpass illumination, (3) white light illumination followed by magnetic actuation and frequency domain filtering, and (4) 635/15nm bandpass illumination followed by magnetic actuation and frequency domain filtering.

Fig. 5. Monochrome images of a 1:1:1 mixture of A-431 cells labeled with 40nm anti-EGFR gold nanoparticles (green arrows), 50nm anti-EGFR gold/iron oxide nanoparticles (red arrows), and unlabeled cells (blue arrows) that were magnetically actuated at 0.9Hz (A) and 1.9Hz (B) before application of a digital frequency filter. The images were obtained using a 635/15nm bandpass filter. Sections (C) and (D) show power spectra that are taken from the time-domain Fourier transform in the region containing a cell labeled with 50nm gold/iron oxide particles (red), 40nm pure gold particles (green) and an unlabeled cell (blue). Note the prominent peaks in the magnetically-labeled cells’ frequency spectra that correspond to the translation stage oscillation frequencies of 0.9Hz (C) and 1.9Hz (D). Sections (E) and (F) show the same fields of view as sections (A) and (B), respectively, after digital filtering at 0.9Hz (E) and 1.9Hz (F) uses the Hanning function implementation. Only magnetically labeled cells are visible.

Results of this analysis are shown in Fig. 6(B) and (C). Under white light illumination, signal from cells labeled with magnetic and non-magnetic gold particles are statistically identical and the unlabeled cells are on average 2.5 times dimmer. Addition of the 635/15nm bandpass filter increases the intensity difference between labeled and unlabeled cells to approximately 4, as indicated in Fig. 6(B). Interestingly, implementation of magnetic actuation and frequency domain filtering leads to statistically the same results independent of which illumination condition is used, as indicated in Fig. 6(C). The average signal intensity ratio between gold/iron oxide and pure gold labeled cells increases from approximately 1 in the case of no magnetic actuation to about 3 with the magnetic actuation. At the same time, the intensity ratio between gold/iron oxide labeled cells and unlabeled cells increases from approximately 2.5–3 to ca. 10 under both illumination conditions. Asterisks in Fig. 6(B) and (C) indicate a statistically significant difference in the average signal values with p<10-4 between the three cell types within each acquisition condition. These results demonstrate that frequency domain filtering is very sensitive to the magnetically controlled movement of cells.

Fig. 6. In (A), pixel intensity profiles are shown for the three cell types: 50nm gold/iron oxide labeled (red line), 40nm pure gold labeled (green line), and unlabeled (blue line). Profiles are drawn for the same three cells captured using white light illumination, 635/15nm bandpass illumination, as well as bandpass plus magnetic actuation and digital frequency filtering. In (B) and (C), the relative average pixel intensity from n>10 cells in each cell population and illumination/acquisition condition is compared. Asterisks and brackets in (B) and (C) indicate a statistical significant difference of the average signal values with p<10-4.

4. Discussion

The use of nanoparticle technology affords a flexible platform for interrogation of biological systems at the molecular level. Remaining barriers exist towards realizing a robust and generalized tool set that could potentially be used in molecular biology and healthcare settings. While issues such as biocompatibility and toxicity are of paramount importance, the ability of nanoparticle-based exogenous contrast agents to generate strong easily detectable signals which are above the endogenous background requires further investigation. For example, in fluorescence imaging techniques, background autofluorescence can present a difficult barrier to overcome [5

X. Gao, Y. Cui, R. M. Levenson, L. W. K. Chung, and S. Nie, “In vivo cancer targeting and imaging with semiconductor quantum dots,” Nature Biotechnology 22, 969–976 (2004). [CrossRef] [PubMed]

]. Further, photobleaching can drastically reduce the ability to monitor longer-term molecular processes or response to therapies. High background scattering from various tissue architecture can make the isolation of molecular specific signals difficult in reflected/scattered light imaging modalities such as reflectance confocal microscopy and OCT. Therefore, the development of multi-faceted approaches is essential to improve the sensitivity of molecular imaging techniques. Here we demonstrate that the combination of plasmon resonance scattering inherent in gold nanoparticles with magnetic actuation results in ca. 10 fold intensity ratio between the labeled and unlabeled cells under white light illumination; this is almost a four times increase over the same ratio for the pure gold nanoparticles. Also, under optimized bandpass illumination the magnetically actuated cells appear 3 times brighter than cells labeled with pure gold nanoparticles after application of the digital filtering, as shown in Figure 6(C). We demonstrated the new approach in A-431 skin cancer cells. These cells are keratinocytes which produce massive amounts of cytokeratin and, therefore, strongly scatter visible light. We chose this unfavorable biological model because of the very high endogenous scattering in order to evaluate the new approach in the presence of a high background signal.

Our results indicate that magnetic actuation of these hybrid nanoparticles may be used to drastically reduce signals from non-magnetically susceptible background sources. This ability may be vital in potential future in-vivo molecular imaging applications, where it is crucial to isolate the distribution of molecules of interest from a dense, highly complex background. Interestingly we note that our results also showed that hybrid magnetic gold nanoparticles can be easily distinguished from pure gold nanoparticles using magnetic actuation. This opens the possibility of a multiplexing approach that uses combinations of magnetic and non-magnetic gold particles in molecularly labeling distinct sub-populations of cells. Further, the analysis algorithm can potentially be incorporated such that Fourier-based filtering can be accomplished in near-real time. There are a number of other potential applications whereby magnetically actuated hybrid particles may be applied. The ability to both magnetically manipulate and monitor cells on the nano-scale with molecular specificity is an exciting direction for further research. For example, pure magnetic nanoparticles (MNPs) were used to probe the mechanical properties of proteins both separately and inside cells [48–50

T. R. Strick, J. F. Allemand, D. Bensimon, A. Bensimon, and V. Croquette, “The elasticity of a single supercoiled DNA molecule,” Science (Washington, D. C.) 271, 1835–1837 (1996). [CrossRef] [PubMed]

]. However, these studies are still limited by micron size of these particles and their relatively low brightness. The magnetically actuated plasmonic nanoparticles can drastically improve the spatial resolution of these studies and signal-to-noise ratio in monitoring of a mechanical response. Further, the field of molecular-specific mechanotransduction may greatly benefit from the ability to both mechanically manipulate cells using an external magnetic field and monitor cellular response in real time with a single agent.

Acknowledgments

We gratefully acknowledge Mr. Kort Travis for nanoparticle scattering and absorption calculations, as well as assistance from Ms. Danielle Smith for X-ray diffraction analysis of gold/iron oxide nanoparticles. Financial support from NCI Grant R01-CA103830-01 BRP is gratefully acknowledged. J. Aaron and S. Kumar were supported by a NSF Integrative Graduate Education and Research Traineeship (IGERT) Grant.

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OCIS Codes
(170.1530) Medical optics and biotechnology : Cell analysis
(170.3880) Medical optics and biotechnology : Medical and biological imaging
(170.4090) Medical optics and biotechnology : Modulation techniques
(170.4580) Medical optics and biotechnology : Optical diagnostics for medicine

ToC Category:
Medical Optics and Biotechnology

History
Original Manuscript: October 18, 2006
Revised Manuscript: December 2, 2006
Manuscript Accepted: December 7, 2006
Published: December 22, 2006

Virtual Issues
Vol. 2, Iss. 1 Virtual Journal for Biomedical Optics

Citation
Jesse S. Aaron, Junghwan Oh, Timothy A. Larson, Sonia Kumar, Thomas E. Milner, and Konstantin V. Sokolov, "Increased optical contrast in imaging of epidermal growth factor receptor using magnetically actuated hybrid gold/iron oxide nanoparticles," Opt. Express 14, 12930-12943 (2006)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-14-26-12930


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References

  1. B. N. G. Giepmans, S. R. Adams, M. H. Ellisman, and R. Y. Tsien, "The Fluorescent Toolbox for Assessing Protein Location and Function," Science (Washington, DC, United States) 312, 217-224 (2006). [CrossRef] [PubMed]
  2. D. J. Bornhop, C. H. Contag, K. Licha, and C. J. Murphy, "Advance in contrast agents, reporters, and detection," J. Biomed. Opt. 6, 106-110. (2001). [CrossRef] [PubMed]
  3. J. Malicka, I. Gryczynski, J. Fang, and R. Lakowicz Joseph, "Fluorescence spectral properties of cyanine dye-labeled DNA oligomers on surfaces coated with silver particles," Analytical Biochemistry 317, 136-146. (2003). [CrossRef] [PubMed]
  4. V. Ntziachristos, C.-H. Tung, C. Bremer, and R. Weissleder, "Fluorescence molecular tomography resolves protease activity in vivo," Nature Medicine (New York, NY, United States) 8, 757-761 (2002). [CrossRef] [PubMed]
  5. X. Gao, Y. Cui, R. M. Levenson, L. W. K. Chung, and S. Nie, "In vivo cancer targeting and imaging with semiconductor quantum dots," Nature Biotechnology 22, 969-976 (2004). [CrossRef] [PubMed]
  6. W. C. Chan, and S. Nie, "Quantum dot bioconjugates for ultrasensitive nonisotopic detection," Science 281, 2016-2018. (1998). [CrossRef] [PubMed]
  7. X. Gao, L. Yang, J. A. Petros, F. F. Marshall, J. W. Simons, and S. Nie, "In vivo molecular and cellular imaging with quantum dots," Current Opinion in Biotechnology 16, 63-72 (2005). [CrossRef] [PubMed]
  8. 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 (Washington, DC, United States) 307, 538-544 (2005). [CrossRef] [PubMed]
  9. A. P. Alivisatos, W. Gu, and C. Larabell, "Quantum Dots as Cellular Probes," inAnnual Review of Biomedical Engineering(2005), pp. 55-76.
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  11. S. J. Oldenburg, R. D. Averitt, S. L. Westcott, and N. J. Halas, "Nanoengineering of optical resonances," Chem. Phys. Lett. 288, 243-247 (1998). [CrossRef]
  12. K. Sokolov, M. Follen, J. Aaron, I. Pavlova, A. Malpica, R. Lotan, and R. Richards-Kortum, "Real-time vital optical imaging of precancer using anti-epidermal growth factor receptor antibodies conjugated to gold nanoparticles," Cancer Research 63, 1999-2004 (2003). [PubMed]
  13. C. Loo, L. Hirsch, M.-H. Lee, E. Chang, J. West, N. Halas, and R. Drezek, "Gold nanoshell bioconjugates for molecular imaging in living cells," Opt. Lett. 30, 1012-1014 (2005). [CrossRef] [PubMed]
  14. I. H. El-Sayed, X. Huang, and M. A. El-Sayed, "Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: Applications in oral cancer," Nano Letters 5, 829-834 (2005). [CrossRef] [PubMed]
  15. K. Sokolov, J. Aaron, B. Hsu, D. Nida, A. Gillenwater, M. Follen, C. MacAulay, K. Adler-Storthz, B. Korgel, M. Descour, R. Pasqualini, W. Arap, W. Lam, and R. Richards-Kortum, "Optical systems for in vivo molecular imaging of cancer," Technology in Cancer Research & Treatment 2, 491-504 (2003). [PubMed]
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