1. Biomedical Engineering; 2. Center for Nano- and Molecular … · 2014-01-05 · Experimental...

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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 Durr 1,2 , BA Holfeld 3 , O Ekici 6 , T Larson 1,2 , DK Smith 2,4 , BA Korgel 2,4 , K Sokolov 2,5 , and A Ben-Yakar 1,2,6,** 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. P in = 0.04 mW 20 μm 20 μm P in = 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. P in = 0.90 mW P in = 0.03 mW P in = 1.13 mW P in = 1.41 mW P in = 1.76 mW P in = 0.04 mW P in = 0.05 mW P in = 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. 0 0.2 0.4 0.6 0.8 1 400 500 600 700 800 900 Wavelength (nm) Absorbance Log(P in (μW)) Log(TPL (arb. units)) 2 IN PL P I 15 nm 50 nm 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/m 2 (right, top), and 50% of isotropically oriented gold nanorods will melt at 30 J/m 2 . This is consistent with experimental results that show no loss in TPL signal at fluences of 5.4 J/m 2 [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. 3.0 4.0 5.0 6.0 7.0 300 500 700 900 1100 1300 1500 gold melting temperature 90% of water critical temperature Temperature, T [K] gold water gold (no HL) Fluence, F [J/m 2 ] XZ reconstruction shows that at large depths, contrast decreases. Cells are resolvable up to depths of approximately 200 μm.

Transcript of 1. Biomedical Engineering; 2. Center for Nano- and Molecular … · 2014-01-05 · Experimental...

Page 1: 1. Biomedical Engineering; 2. Center for Nano- and Molecular … · 2014-01-05 · Experimental I.TPL imaging setup We use a custom-built multiphoton microscope for TPL imaging. The

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.

0

0.2

0.4

0.6

0.8

1

400 500 600 700 800 900

Wavelength (nm)

Ab

so

rban

ce

Log(Pin(μW))

Lo

g(T

PL

(arb

. u

nit

s))

2

INPL PI

15 nm

50 nm

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.

3.0 4.0 5.0 6.0 7.0

300

500

700

900

1100

1300

1500 gold

melting temperature

90% of water

critical temperature

Tem

pera

ture

, T

[K

]

Fluence, F [J/m2]

gold

water

gold (no HL)

3.0 4.0 5.0 6.0 7.0

300

500

700

900

1100

1300

1500 gold

melting temperature

90% of water

critical temperature

Tem

pera

ture

, T

[K

]

Fluence, F [J/m2]

gold

water

gold (no HL)

XZ reconstruction shows that at large depths,

contrast decreases. Cells are resolvable up to

depths of approximately 200 μm.