Combined photothermal therapy and magneto-motive ultrasound imaging using multifunctional nanoparticles

Mohammad Mehrmohammadi1, Li L. Ma2, Yun-Sheng Chen1, Min Qu1, Pratixa Joshi1, Raeanna M. Chen1, Keith P. Johnston2 and Stanislav Emelianov1

1Department of Biomedical Engineering and 2Department of Chemical Engineering University of Texas at Austin, Austin, TX 78712

emelian@mail.utexas.edu

ABSTRACT

Photothermal therapy is a laser-based non-invasive technique for cancer treatment. Photothermal therapy can be enhanced by employing metal nanoparticles that absorb the radiant energy from the laser leading to localized thermal damages. Targeting of nanoparticles leads to more efficient uptake and localization of photoabsorbers thus increasing the effectiveness of the treatment. Moreover, efficient targeting can reduce the required dosage of photoabsorbers; thereby reducing the side effects associated with general systematic administration of nanoparticles. Magnetic nanoparticles, due to their small size and response to an external magnetic field gradient have been proposed for targeted drug delivery. In this study, we investigate the applicability of multifunctional nanoparticles (e.g., magneto-plasmonic nanoparticles) and magneto-motive ultrasound imaging for image-guided photothermal therapy. Magneto-motive ultrasound imaging is an ultrasound based imaging technique capable of detecting magnetic nanoparticles indirectly by utilizing a high strength magnetic field to induce motion within the magnetically labeled tissue. The ultrasound imaging is used to detect the internal tissue motion. Due to presence of the magnetic component, the proposed multifunctional nanoparticles along with magneto-motive ultrasound imaging can be used to detect the presence of the photo absorbers. Clearly the higher concentration of magnetic carriers leads to a monotonic increase in magneto-motive ultrasound signal. Thus, magneto-motive ultrasound can determine the presence of the hybrid agents and provide information about their location and concentration. Furthermore, the magneto-motive ultrasound signal can indicate the change in tissue elasticity – a parameter that is expected to change significantly during the photothermal therapy. Therefore, a comprehensive guidance and assessment of the photothermal therapy may be feasible through magneto-motive ultrasound imaging and magneto-plasmonic nanoparticles.

Keywords: Photothermal therapy, magneto-motive ultrasound, magneto-plasmonic nanoparticles, hybrid nanoparticles

1. INTRODUCTION Photothermal therapy (PT therapy) is a non-invasive, laser-based technique with many clinical applications including cancer treatment. During photothermal therapy, light energy is converted to heat within the tissue causing the thermal destruction of tissue within the tumor. Therefore, it is very critical to selectively heat the diseased tissue (i.e., cancerous cells) to achieve the tumor necrosis without affecting the surrounding normal tissue.[1, 2] Unfortunately, there may not a significant contrast between the light absorption in normal and cancerous tissue. In addition, high laser fluence may be needed to sufficiently heat deeply embedded tumors leading to thermal damage in the intervening tissue between the cancer site and the laser source. Therefore, there is a need for an agent that is absorbing the light at the wavelengths where tissue does not show significant absorption.[3, 4] The size of the agents should be small enough to be delivered to the diseased cells via intravenous injection. Metal-based plasmonic nanoparticles are being extensively used as molecular specific contrast agents for various imaging and therapeutics applications.[5, 6] They are biocompatible, can be delivered to the diseased tissue region, efficiently absorb the laser irradiation and transfer the heat to their surrounding tissue.[7] Furthermore, the nanoparticles can be tuned to have plasmon-resonance in the near infra-red (NIR) spectrum by varying the geometry (size, aspect ratio, etc.)[8] or using nano-composite materials.[9]

Nanoscale Imaging, Sensing, and Actuation for Biomedical Applications VII, edited by Alexander N. Cartwright, Dan V. Nicolau, Proc. of SPIE Vol. 7574, 757405

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The efficiency of the nanoparticle-assisted PT therapy is directly related to the presence and accumulation of the nanoparticles in the tumor. Clearly, higher selective accumulation can lead to larger heat generation and thus more efficient therapeutic procedure. Moreover, to execute photothermal therapy successfully, there is a need for a diagnostic imaging technique to identify the size and spatial location of the diseased tissue (i.e., tumor) and to monitor delivery and accumulation of nanoparticles within the tissue. Finally, assessment of the therapy outcome is very important. In other words, thermal damage must be quantitatively evaluated to ensure complete tumor destruction. In this study we demonstrate the potential application of magneto-plasmonic nanoparticles for efficient and controlled PT therapy applications.

Nanoclusters containing gold (Au) and iron-oxide (Fe3O4) are an example of such magneto-plasmonic nanoparticles (Figure 1). These nanoclusters are formed by the reduction of HAuCl4 onto the surfaces of 5 nm iron oxide nanoparticles with hydroxylamine as a seeding agent. The hydroxylamine is adsorbed on the iron oxide particle surface and favors selective formation of gold on the iron oxide surface rather than in bulk solution.[10] The dextran molecules on the iron oxide surface help to prevent the gold domains from growing too thick during reduction. The reaction resulted in ~30 nm relatively open clusters composed of much smaller primary particle domains. The primary domains are easily discerned near the periphery in the TEM images (Figure 1a). The small hydrodynamic diameter of the nanorose in DI water ranged from 23±3 and 34±2. The total polymer concentration was less than 12% according to thermal gravimetric analysis (TGA), in contrast with larger amounts typically needed in block copolymer templated clusters.[11] An energy-dispersive X-ray spectroscopy (EDS) area scan across the particle shows that iron was present throughout the cluster (Figure 1b).

Au Fe

a

c d

b

AuAu Fe

a

c d

b

Figure 1. a) High-resolution transmission electron microscopy (TEM) image of an Au-Fe3O4 nanocluster (nanorose), b) EDS

area mapping of Au-Fe3O4 nanocluster, c) UV-Vis-NIR absorbance spectra of colloidal dispersion of nanoclusters in various media (DI water, 1X PBS solution, and a DMEM supplemented with 10% FBS cell culture media), and d) normalized magnetization of nanoclusters per gram of Fe3O4 measured at 300 K.

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The broad UV-Vis-NIR absorbance of a colloidal dispersion of nanoclusters is shown in Figure 1c. Colloidal and optical stability of the nanoclusters may be attributed to prevention against growth or collapse of the gold domains by the iron oxide cores and polymer stabilizers for both the primary domains and the external surfaces of the clusters. The nanorose extinction cross-section at 755 nm is 6 orders of magnitude larger than that of freshly prepared indocyanine green dissolved in NaCl aqueous buffer solution (1·10-20 m2 at 778 nm), which has been investigated as a NIR dye for laser photothermal therapy.[12, 13] The normalized saturation magnetization at 300 K (Figure 1d) was 34 emu/g Fe3O4 as measured by a superconducting quantum interference device (SQUID) magnetometer (Quantum Design MPMS). To convert the magnetization per total mass of particles to a basis of per mass of Fe3O4, the 3:1 mass ratio of Au/Fe, determined by flame atomic absorption spectroscopy (FAAS), and the 12% polymer amount, determined by TGA, was used. The magnetization approached the value of 39 emu/g Fe3O4 (54 emu/g Fe) for the original 5 nm iron oxide nanoparticles, indicating little interference from the gold coating. [14]

The magneto-plasmonic nanoparticles are characterized by both optical absorption and magnetic susceptibility. The absorption in near infra-red (NIR) spectrum is attributed to the presence of gold in their composition while the magnetic susceptibility comes from the presence of magnetite in the hybrid nanoparticle. In this study, we investigate the applicability of magneto-motive ultrasound imaging and multifunctional nanoparticles (e.g., magneto-plasmonic nanoparticles) for image-guided photothermal therapy. Due to magnetic susceptibility of magneto-plasmonic nanoparticles, their accumulation at the therapeutic site can be significantly increased by applying an external magnetic field using, for example, a permanent magnet. We also introduce pulsed magneto-motive ultrasound (PMMUS) imaging technique capable of in-vivo imaging of magnetic nanoparticles in real-time and at sufficient depth.[15] PMMUS imaging can potentially be used to detect the presence and to assess the accumulation of hybrid magneto-plasmonic nanoparticles in tissue. Finally, PMMUS imaging can also be used to determine the outcome of the therapy procedure since the magneto-motive ultrasound signal can indicate the change in tissue elasticity – a parameter that is expected to change significantly during the photothermal therapy. Therefore, a comprehensive guidance and assessment of the photothermal therapy may be feasible using magneto-motive ultrasound imaging and magneto-plasmonic nanoparticles.

2. MATERIALS AND METHODS In this study, three different sets of experiments were performed to demonstrate the potential of magneto-plasmonic nanoparticles in image-guided photothermal therapy. Specifically, our study was designed to demonstrate 1) enhanced accumulation of magneto-plasmonic nanoparticle in tissue produced by application of external magnetic field, 2) remote assessment of the magneto-plasmonic nanoparticle accumulation within the tissue using PMMUS imaging, and 3) assessment of the outcome of photothermal treatment using PMMUS sensing of tissue elasticity change.

2.1 Magneto-enhanced delivery of the magnetic agents

The idea of using magnetic nano-carriers to target specific sites in the body dates back to the late 1970s.[16, 17] By placing a high-gradient magnetic field (i.e., a strong permanent magnet) at the tissue surface, the interaction between the magnetic field and the magnetic nanoparticles can enhance the accumulation of nanoparticles within the desired region of tissue (i.e., tumor). To demonstrate this effect, we performed an initial in-vivo study using two tumor bearing Nu/Nu mice. Both animals were subcutaneously inoculated with 1·106 (100-µl injection volume) A431 human epithelial carcinoma cells (American Type Culture Collection, VA). When the tumors reached 6-8 mm in diameter, the 5 nm superparamagnetic iron-oxide (SPIO) nanoparticles (Feridex I.V., Bayer Healthcare, Inc.) were administered intravenously in the blood stream through tail vein injection. The same dose of magnetic nanoparticles (1 mmol/kg Fe3O4) was injected into both animals. A strong neodymium cylindrical disk magnet with nominal axial magnetization of ~0.64 T was placed close to the tumor in one of the animals and another animal without magnet was used as a control. Both mice were euthanized 24 hours after the injection of nanoparticles and the tumors were harvested and fixed in a formalin. Histological evaluation (Prussian blue staining) was performed to evaluate the accumulation of iron-oxide nanoparticles in tumor.

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2.2 Pulsed magneto-motive ultrasound (PMMUS) imaging of magnetic nanoparticles

Pulsed magneto-motive ultrasound (PMMUS) is an ultrasound-based imaging technique, capable of imaging magnetic nanoparticles indirectly.[15] The significant difference between magnetic susceptibility of normal tissue and the magnetic nanoparticles is the basis of the contrast mechanism in pulsed magneto-motive ultrasound imaging. PMMUS utilizes a high strength pulsed magnetic field to induce motion within the magnetically labeled tissue and ultrasound to detect the internal tissue motion.

To demonstrate the ability of the PMMUS imaging system to detect the magnetic nanocomposites within tissue, a set of experiments was performed on a sample of porcine longissimus dorsi muscle with thickness of approximate 15 mm injected with plain and magnetically labeled macrophage cells suspended within 12% gelatin. The mouse monocytes – macrophages cells (J 774 A.1 cell line) with a high rate of non-specific uptake were cultured in DMEM, supplemented with 5% FBS at 37º C in a 5% CO2 environment. To magnetically label cells, macrophages were incubated in a suspension of 50 nm carbon-coated cobalt nanoparticles (TurboBeads Inc., Switzerland) solution diluted to 0.5 mgr/ml of Co concentration. After 24 hours, cells were harvested and mixed with 10% gelatin and 1% of silica microparticles (acting as ultrasound scatterers). The mixture at 35ºC temperature was injected into the porcine muscle sample, and the sample was then cooled down to allow gel to harden. To have a control sample, an inclusion of 12% gelatin and non-labeled macrophages cells (i.e., cell with no magnetic nanoparticles) was also injected into the same tissue sample.

Figure 2. Diagram of a custom-built PMMUS imaging system

The PMMUS imaging system, used in this study, consisted of a magnetic field generator interfaced with an ultrasound imaging system. A block diagram of the custom-built PMMUS imaging system is shown in Figure 2. A focused high-frequency single-element ultrasound transducer (7.5 MHz center frequency, focal depth = 50.8 mm, f-number = 4) was used to capture ultrasound RF data. The magnetic field generator was based on a commercially available magnetic pulser (MPG5, SOTA, Inc.) but modified to meet the imaging system requirements. An iron-core was embedded into the coil in order to focus and maximize the magnetic field in the desired imaging region. The magnetic pulse strength, measured 5 mm above the iron-core tip using a digital gaussmeter (DSP 475, Lakeshore Inc.), was 0.6 Tesla. Prior to imaging

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experiment, the focal point of ultrasound transducer and the iron-core tip were axially aligned. To obtain the images, the mechanical scanning of the tissue sample with lateral step of 200 µm was performed using a 2-D motion axes (x-y plane) attached to the water cuvette while the ultrasound transducer and the magnetic coil were kept fixed. A microprocessor-based unit was utilized to control the positioning system, the ultrasound pulser-receiver, and the data acquisition unit. The cross-correlation motion tracking technique[18] was used to assess the tissue displacement induced by the external magnetic field. Both ultrasound B-scans and magneto-motive ultrasound images were reconstructed off-line. The pulsed magneto-motive ultrasound imaging system was designed to capture several pulse-echo signals before the application of the magnetic pulse. The pre-excitation RF lines were used as the reference to compute the relative displacement during the pulsed magnetic excitation as well as to reconstruct the B-scan ultrasound image. The relatively short magnetic pulses (6-10 ms) caused the iron-laden tissue to move and to reach the maximum displacement in about 50 ms after the pulse. The high pulse repetition frequency of ultrasound pulse-echo imaging was used to insure reliable measurement of the tissue displacement. The gross motion of the sample and water cuvette was eliminated by using the bottom of the water cuvette as a stationary reference. The maximum displacement at each position within the imaging plane was then used to form the magneto-motive ultrasound image. 2.3 PMMUS sensing of tissue elasticity to assess the outcome of the photothermal therapy

The viscoelastic properties of healthy tissue, cancerous tissue and thermally damaged tissue are vastly different.[19, 20] Therefore, assessment of the tissue elasticity can be used as a tool to assess the effectiveness of the photothermal treatment. Tissue elasticity plays an important role in magnetically induced tissue displacement during the PMMUS imaging. Therefore, pulsed magneto-motive ultrasound offers an interesting potential ability for elasticity imaging. To demonstrate the capability of PMMUS imaging system to assess the elasticity of tissue, a set of experiments were performed where the magnetic inclusions (8% gelatin mixed with 5 nm SPIO suspensions at the concentration of 0.5 mgr/ml Fe) were embedded into a 3% gelatin background. Since the shear modulus of the gelatin is decreasing with temperature, the temperature of the phantom was varied in the 12°C (stiffer background) to 22°C (softer background) to change the elasticity of the background [21] while the PMMUS imaging was performed at different temperatures.

3. RESULTS AND DISCUSSION Figure 3 shows the histological slices of the tumors from the animals injected with magnetic nanoparticles and kept for 24 hours without (left) and with (right) the permanent magnet adjacent to skin above the tumor. The blue regions indicate the presence of the iron-oxide magnetic nanoparticles. Clearly, the magnetic targeting enhanced the accumulation of nanoparticles. Although the accumulation of magnetic nanoparticles within the tumors depends on many parameters such as the stage of tumor development, the location of the tumor, tumor type, etc., our study primarily indicate the effect of the magneto-enhanced delivery of magneto-plasmonic nanoparticles. Therefore, hybrid nanoparticles possessing both magnetic susceptibility and optical absorption are suitable candidate for magnetic delivery of NIR absorbers to the tumor. Once the higher concentration of magneto-plasmonic therapeutic agent is delivered to the tumor, the photothermal therapy can be performed more efficiently.

Figure 3. Histological assessment (Prussian blue staining) of the control tumor (left) and tumor with magneto-enhanced

delivery of magnetic nanoparticles.

Figure 4 shows the results of PMMUS imaging of magnetically labeled macrophage cells. Although the ultrasound B-scan (Fig. 4a) identifies the location of the gelatin inclusions within the background tissue, it is the MMUS image that

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clearly detects the presence of magnetically labeled cells within the tissue background (Fig. 4b). The porcine tissue has negligible magnetic susceptibility compared to the inclusion with magnetically labeled cells. The low intensity of ultrasound signal below the inclusions is due to high concentration of ultrasound scatter agents (silica microparticles) within the inclusions. The magneto-motive ultrasound image has high contrast between the magnetic inclusion and control inclusion based on the presence of magnetically labeled tissue. Given the mechanical continuum, there is a small motion of the background tissue near the inclusion and in the control inclusion – this background motion is more likely induced by the motion within the magnetically labeled inclusion. The average displacement in the inclusion region was about 50 µm. Therefore, these results suggest that PMMUS imaging has a sensitivity to localize the magnetic nanoparticles in tissue.

1 mm

ba

Plain cells Magnetically labeled cells

1 mm1 mm

ba

Plain cells Magnetically labeled cells Figure 4. (a) Ultrasound B-scan and (b) MMUS image of the porcine tissue injected with unlabeled (i.e., native) and

magnetically labeled cell.

The MMUS displacement of the magnetically labeled tissue and the shear modulus of tissue are inversely proportional.[22] Therefore, assuming the constant concentration of magnetic nanoparticles during the photothermal therapy, changes in tissue motion may be indicative of tissue elasticity. The displacement of the magnetic inclusion embedded within the gelatin background with different shear modulus is shown in Figure 5. As expected, there is a monotonic decrease of the detected displacement as the shear modulus of the background increases. Similar behavior is expected during the photothermal therapy where tissue elasticity will increase during and after a successful photothermal therapy procedure. Therefore, PMMUS can serve as an imaging tool to assess the tissue elasticity and thus to assess the outcome of photothermal therapy.

Det

ecte

d di

spla

cem

ent (

µm)

Elasticity (kPa)

Det

ecte

d di

spla

cem

ent (

µm)

Elasticity (kPa) Figure 5. (b) MMUS displacement of magnetically labeled tissue with different shear elasticity.

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4. CONCLUSION In summary, the feasibility of magneto-motive ultrasound imaging and magneto-plasmonic nanoparticles for high-efficiency and controlled photothermal therapy procedure was initially investigated. Pulsed magneto-motive ultrasound imaging was introduced as an imaging modality capable of detecting magnetically labeled tissue and, therefore, assessing of the accumulation of the photothermal therapy agent within the therapeutic region. Moreover, the spatio-temporal dynamic behavior of magnetically labeled tissue is related to viscoelastic properties of the tissue and, therefore, MMUS imaging during photothermal therapy can be used to remotely palpate the tissue thus confirming the effectiveness of the photothermal therapy.

5. ACKNOWLEDGMENTS The authors are grateful to Dr. Konstantin Sokolov and Ms. Kimberly Homan of the University of Texas at Austin for the helpful discussions on characterization of nanoparticles. The authors also would like to thank Dr. Salavat Aglyamov and Dr. Andrei Karpiouk of the University of Texas at Austin for their help in MMUS elasticity measurements and discussion of the results. This work was partially supported by the National Institutes of Health under grant EB 008821.

REFERENCES [1] M. H. Falk and R. D. Issels, "Hyperthermia in oncology," Int J Hyperthermia 17, 1-18 (2001). [2] S. N. Goldberg, G. S. Gazelle, and P. R. Mueller, "Thermal ablation therapy for focal malignancy: a unified

approach to underlying principles, techniques, and diagnostic imaging guidance," AJR Am J Roentgenol 174, 323-31 (2000).

[3] M. A. El-Sayed, I. H. El-Sayed, W. Qian, and X. Huang, "Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods," Journal of the American Chemical Society 128, 2115 (2006).

[4] J. Shah, S. Park, S. Aglyamov, T. Larson, L. Ma, K. Sokolov, K. Johnston, T. Milner, and S. Y. Emelianov, "Photoacoustic imaging and temperature measurement for photothermal cancer therapy," J Biomed Opt 13, 034024 (2008).

[5] P. Debbage and W. Jaschke, "Molecular imaging with nanoparticles: giant roles for dwarf actors," Histochem Cell Biol 130, 845-75 (2008).

[6] G. Kong, R. D. Braun, and M. W. Dewhirst, "Hyperthermia enables tumor-specific nanoparticle delivery: effect of particle size," Cancer Res 60, 4440-5 (2000).

[7] C. Loo, A. Lin, L. Hirsch, M. H. Lee, J. Barton, N. Halas, J. West, and R. Drezek, "Nanoshell-enabled photonics-based imaging and therapy of cancer," Technol Cancer Res Treat 3, 33-40 (2004).

[8] S. Link and M. A. El-Sayed, "Spectral properties and relaxation dynamics of surface plasmon electronic oscillations in gold and silver nanodots and nanorods," J. phys. chem. B 103, 8410-8426 (1999).

[9] C. Loo, A. Lowery, N. Halas, J. West, and R. Drezek, "Immunotargeted nanoshells for integrated cancer imaging and therapy," Nano Lett 5, 709-11 (2005).

[10] 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 Lett 4, 719-723 (2004).

[11] Y. Ofir, B. Samanta, and V. M. Rotello, "Polymer and biopolymer mediated self-assembly of gold nanoparticles," Chem Soc Rev 37, 1814-25 (2008).

[12] W. R. Chen, R. L. Adams, R. Carubelli, and R. E. Nordquist, "Laser-photosensitizer assisted immunotherapy: a novel modality for cancer treatment," Cancer letters 115, 25-30 (1997).

[13] K. Urbanska, B. Romanowska-Dixon, Z. Matuszak, J. Oszajca, P. Nowak-Sliwinsk, and G. Stochel, "Indocyanine green as a prospective sensitizer for photodynamic therapy of melanomas," Acta Biochimica Polonica 49, 387 (2002).

[14] L. L. Ma, M. D. Feldman, J. M. Tam, A. S. Paranjape, K. K. Cheruku, T. A. Larson, J. O. Tam, D. R. Ingram, V. Paramita, J. W. Villard, J. T. Jenkins, T. Wang, G. D. Clarke, R. Asmis, K. Sokolov, B. Chandrasekar, T. E. Milner, and K. P. Johnston, "Small multifunctional nanoclusters (nanoroses) for targeted cellular imaging and therapy," ACS Nano 3, 2686-96 (2009).

Proc. of SPIE Vol. 7574 757405-7

Downloaded from SPIE Digital Library on 15 Dec 2010 to 128.83.154.154. Terms of Use: http://spiedl.org/terms

[15] M. Mehrmohammadi, J. Oh, S. R. Aglyamov, A. B. Karpiouk, and S. Y. Emelianov, "Pulsed magneto-acoustic imaging," Conf Proc IEEE Eng Med Biol Soc 1, 4771-4 (2009).

[16] K. Mosbach and U. Schröder, "Preparation and application of magnetic polymers for targeting of drugs," FEBS letters 102, 112 (1979).

[17] A. Senyei, K. Widder, and G. Czerlinski, "Magnetic guidance of drug carrying microspheres," Journal of Applied Physics 49, 3578 (1978).

[18] N. A. Cohn, S. Y. Emelianov, M. A. Lubinski, and M. O'Donnell, "An elasticity microscope. Part I: methods," IEEE Transactions on Ultrasonics, Ferroelectrics and Frequency Control 44, 1304-1319 (1997).

[19] A. Sarvazyan, "Mechanical imaging: a new technology for medical diagnostics," Int J Med Inform 49, 195-216 (1998).

[20] T. Varghese, J. A. Zagzebski, and F. T. Lee, Jr., "Elastographic imaging of thermal lesions in the liver in vivo following radiofrequency ablation: preliminary results," Ultrasound Med Biol 28, 1467-73 (2002).

[21] A. B. Karpiouk, S. R. Aglyamov, Y. A. Ilinskii, E. A. Zabolotskaya, and S. Y. Emelianov, "Assessment of shear modulus of tissue using ultrasound radiation force acting on a spherical acoustic inhomogeneity," IEEE Trans Ultrason Ferroelectr Freq Control 56, 2380-7 (2009).

[22] A. Oldenburg, F. Toublan, K. Suslick, A. Wei, and S. Boppart, "Magnetomotive contrast for in vivo optical coherence tomography," Opt Express 13, 6597-614 (2005).

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