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1. Biomedical Engineering; 2. Center for Nano- and Molecular … · 2014-01-05 · Experimental...
Transcript of 1. Biomedical Engineering; 2. Center for Nano- and Molecular … · 2014-01-05 · Experimental...
Experimental
I.TPL imaging setup We use a custom-built multiphoton microscope
for TPL imaging. The setup delivers 300 fs pulses
to the sample via galvanometric scanning mirrors
and a 0.95 NA water-dipping objective lens.
II. Gold Nanorod Synthesis Gold nanorods were synthesized using a seed
mediated growth method, starting with ~1.5 nm
diameter colloidal gold seeds and using CTAB as
a surfactant. [3]
III. Conjugation and Tissue Phantom
Preparation The positive surface potential from the CTAB was
changed to negative by coating the CTAB with
PSS. [4] Anti-EGFR antibodies were mixed and
bound to form the molecularly targeted nanorod
contrast agent.
Two-photon Luminescence Imaging Using Gold Nanorods as Bright Contrast Agents NJ Durr1,2, BA Holfeld3, O Ekici6, T Larson1,2, DK Smith2,4, BA Korgel2,4, K Sokolov2,5, and A Ben-Yakar1,2,6,**
1. Biomedical Engineering; 2. Center for Nano- and Molecular Science and Technology; 3. Physics; 4. Chemical Engineering, Texas Materials Institute,
5. University of Texas M.D. Anderson Cancer Center; 6. Mechanical Engineering; **Contact: [email protected] web: www.me.utexas.edu/ben-yakar/
The University of Texas at Austin
Introduction I. Two-photon Microscopy for Diagnostic Imaging More than 85% of all cancers begin as precancerous lesions that
are confined to the surface epithelium, which can be many
hundreds of microns thick in human tissue. Two-photon imaging
(TPI) is a powerful technique for the early diagnosis of epithelial
cancers because it permits real time, three-dimensional, non-
invasive, molecular imaging hundreds of microns deep into tissue.
Morphological and fluorescence quantification from TPI of
endogenous fluorophores can be used to distinguish cancerous
and precancerous from normal tissue.
Results I. We observe a three orders of magnitude increase in
emission signal of nanorod labeled cancer cells
compared to unlabeled cancer cells.
II. Bright contrast was achieved with negligible increase
in bulk scattering coefficient in a tissue phantom
III. TPL Imaging was performed
down to 200μm deep in a tissue
phantom.
Acknowledgments We acknowledge Quoc Nguyen for providing the source code for his MPScan program, and Pengyuan
Chen and Benny Hwang for their assistance in modifying the program for use in our microscope. D.S.
and B.K. acknowledge partial funding of this research by The Robert A. Welch Foundation, the
National Institutes of Health and the National Science Foundation (CHE-9876674). A.B. and N.D.
acknowledge support by the National Institute of Health NIH Grant RO3 CA125774-01 and National
Science Foundation Grants BES-0548673 and BES-0508266. N.D. acknowledge support by a National
Science Foundation IGERT Fellowship.
References 1. N.J. Durr, et al, ““Two-photon luminescence imaging of cancer cells using molecularly targeted gold
nanorods,” Nano Letters, vol. 7, Apr. 2007, pp. 941-945.
2. H. Wang, et al, “In vitro and in vivo two-photon luminescence imaging of single gold nanorods”,
PNAS (2), 15752-6 (2005)
3. N. R. Jana, et al, “Seed-Mediated Growth Approach for Shape-Controlled Synthesis of Spheroidal
and Rod-like Gold Nanoparticles Using a Surfactant Template”, Adv Mat (13), 1389-93 (2001)
4. X. Huang, et al, “Cancer Cell Imaging and Photothermal Therapy in the Near-Infrared Region by
Using Gold Nanorods”, JACS (128), 2115-20 (2006)
III. Nanorods as Two-Photon Contrast Agents Metallic nanorods are attractive two-photon contrast agents [1,2]
when compared to traditional organic fluorescent contrast agents
and quantum dots primarily because they exhibit three favorable
properties:
1. Biocompatibility
2. Brightness
3. Photostability
3D rendering of healthy human tongue biopsy imaged 240μm deep. Left: rendering with
all images. Right: rendering with top half of images removed to visualize epithelial
structure. Red channel: λ=450-680nm emission autofluorescence. Green channel:
λ=430nm emission second harmonic generation. The basal layer can be distinguished
by the barrier between the green and red signals.
Left: Two-photon autofluorescence image of unlabeled A431 human epithelial cancer cell phantom; Middle: SEM
image of 50 nm x 15 nm gold nanorods; Right: Two-photon luminescence image of labeled A431 cell phantom.
II. Two-Photon Luminescence Imaging Metallic particles reduced to the nanometer size can exhibit surface
plasmon resonance. Nanorods can be tuned so that their resonance
is at frequencies corresponding to the near-infrared light we use as
an excitation source. At this resonance frequency, the nanorods
interact strongly with light, allowing for appreciable absorption of
near-simultaneous photons at high intensities. With our nanorods,
we observe a two photon process, as evidenced by the quadratic
dependence of emission intensity on excitation intensity.
Left: Absorbance from various shaped nanorods. Middle: Quadratic dependence of
emission intensity on excitation power. Right: Overlay of TPL emission intensity,
absorbance, and excitation spectrum.
Pin= 0.04 mW 20 μm 20 μm Pin= 1.13 mW
Left: Two-photon autofluorescence image of unlabeled cells at high power.
Right: Two-photon luminescence image of gold nanorod labeled cells at 64x less
power. This corresponds to a greater than 4,000x brighter emission signal from
the nanorods than autofluorescence.
10 μm
Two-photon luminescence images of nanorod labeled cells at z = 10
(left), 14 (middle), and 18 (right) μm
Two-photon imaging of cancer cells embedded in a collagen matrix at increasing
depths. (a) TPAF imaging of unlabeled cells and (b) TPL imaging of nanorods
labeled cells. The power was increased by 26 % at each 20 μm depth increment to
maintain constant emission intensity collection.
Pin= 0.90 mW
Pin= 0.03 mW
Pin= 1.13 mW Pin= 1.41 mW Pin= 1.76 mW
Pin= 0.04 mW Pin= 0.05 mW Pin= 0.07 mW
Abstract We demonstrate the use of gold nanorods as bright contrast agents for two-photon
luminescence (TPL) imaging of cancer cells in a three dimensional tissue phantom at depths
of up to 150 μm. The TPL intensity from gold nanorod labeled cancer cells can be three orders
of magnitude brighter than the two-photon autofluorescence (TPAF) emission intensity from
unlabeled cancer cells at 760 nm excitation light [1]. Their strong signal, resistance to
photobleaching, chemical stability, ease of synthesis, simplicity of conjugation chemistry, and
biocompatibility make gold nanorods an attractive contrast agent for cancer diagnostics using
TPL imaging.
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400 500 600 700 800 900
Wavelength (nm)
Ab
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Anti-EGFR conjugated PSS Coated CTAB Coated
Modeling Gold nanorods exposed to femtosecond pulses will melt above a certain energy threshold. To determine this threshold, we constructed a two-temperature computational model. We calculate that a gold nanorod aligned to the pulse polarization will melt at 7 J/m2 (right, top), and 50% of isotropically oriented gold nanorods will melt at 30 J/m2. This is consistent with experimental results that show no loss in TPL signal at fluences of 5.4 J/m2 [1]. The temperature profile of a gold nanorod 70ps after irradiation is shown at the bottom right. No significant energy accumulation between consecutive pulses is found for typical imaging fluences.
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melting temperature
90% of water
critical temperature
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Fluence, F [J/m2]
gold
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gold (no HL)
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melting temperature
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critical temperature
Tem
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Fluence, F [J/m2]
gold
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gold (no HL)
XZ reconstruction shows that at large depths,
contrast decreases. Cells are resolvable up to
depths of approximately 200 μm.