nihms119544.pdf

In vivo

carbon nanotube-enhanced non-invasive photoacoustic

mapping of the sentinel lymph node

Manojit Pramanik

Kwang Hyun Song

Magdalena Swierczewska

Danielle Green

Balaji

Sitharaman

, and

Lihong V. Wang

Optical Imaging Laboratory, Department of Biomedical Engineering, Washington University in St.

Louis, Missouri 63130, USA

Department of Biomedical Engineering, State University of New York at Stony Brook, Stony Brook,

New York 11794, USA

Abstract

Sentinel lymph node biopsy (SLNB), a less invasive alternative to axillary lymph node dissection

(ALND), has become the standard of care for patients with clinically node-negative breast cancer.

In SLNB, lymphatic mapping with radio-labeled sulfur colloid and/or blue dye helps identify the

sentinel lymph node (SLN), which is most likely to contain metastatic breast cancer. Even though

SLNB, using both methylene blue and radioactive tracers, has a high identification rate, it still relies

on an invasive surgical procedure, with associated morbidity. In this study, we have demonstrated a

non-invasive single-walled carbon nanotube (SWNT)-enhanced photoacoustic (PA) identification

of SLN in a rat model. We have successfully imaged the SLN

in vivo

by PA imaging (793 nm laser

source, 5 MHz ultrasonic detector) with high contrast-to-noise ratio (= 89) and good resolution (~500

m). The SWNTs also show a wideband optical absorption, generating PA signals over an excitation

wavelength range of 740–820 nm. Thus, by varying the incident light wavelength to the near infrared

region, where biological tissues (hemoglobin, tissue pigments, lipids, and water) show low light

absorption, the imaging depth is maximized. In the future, functionalization of the SWNTs with

targeting groups should allow the molecular imaging of breast cancer.

1. Introduction

For the majority of invasive breast cancers, the surgical removal of primary breast tumor and

level I and level II axillary lymph node dissections (ALND) are widely performed (NIH

Consensus Statement 1990). However, the common side effects after ALND include upper-

extremity lymphedema, arm numbness, impaired shoulder mobility, arm weakness, and

infections in the breast, chest, or arm (Swenson

et al

2002). A less invasive, more accurate

alternative to ALND is sentinel lymph node biopsy (SLNB). For patients with clinically node-

negative breast cancer, SLNB has rapidly become the standard of care (Krag

et al

1998,

McMasters

et al

2000). The concept of a sentinel lymph node biopsy assumes that the primary

draining or sentinel node will be the first to contain metastases. The hypothesis is that both the

mammary gland and overlying skin share a common lymphatic pathway to the same axillary

sentinel node. Therefore, intradermal injection of blue dye will lead to the accumulation of the

dye in the sentinel lymph nodes (

Borgstein

et al

1997

). In this surgery, a special blue dye and/

or a radioactive substance is first injected into the breast to determine which lymph nodes are

the first to receive drainage from the breast. These nodes are potentially the first to be invaded

by cancer cells. One to three sentinel nodes are usually removed and tested for cancer. If

E-mail: balaji.sitharaman@stonybrook.edu (CNT), lhwang@biomed.wustl.edu (PA).

NIH Public Access

Author Manuscript

Phys Med Biol

. Author manuscript; available in PMC 2009 August 26.

Published in final edited form as:

Phys Med Biol

. 2009 June 7; 54(11): 3291–3301. doi:10.1088/0031-9155/54/11/001.

NIH-PA Author Manuscript

cancerous, then all the lymph nodes are removed. This surgery has fewer complications than

axillary node dissection, but the physicians performing the procedure must have special

training. The identification rate and sensitivity of this technique are less than 95%, even in

experienced hands (Krag

et al

1998, McMasters

et al

2000, Ung 2004). Furthermore, the

complications associated with the SLNB procedure include seroma formation, lymphedema,

sensory nerve injury, and limitation in the range of motion (Purushotham

et al

2005). These

limitations of SLNB strongly suggest that alternative strategies to stage the axilla should be

explored.

Axillary ultrasound (AUS) has been proposed as a potential non-invasive technique for

identifying axillary metastases (Krishnamurthy

et al

2002, Deurloo

et al

2003, Brancato

2004). AUS can visualize the lymph node’s size, shape, and contour, as well as changes in

cortical morphology and texture that appear to be associated with the presence of axillary

metastases. However, the ability of AUS alone to stage the axilla accurately is limited because

the sonographic signs of metastatic disease may overlap with those of benign reactive changes.

On the other hand,

in vivo

identification of a sentinel lymph node (SLN) using photoacoustic

(PA) imaging would allow non-invasive axillary staging, in conjunction with either

percutaneous fine needle aspiration biopsy (FNAB) or other emerging molecular techniques.

Photoacoustic tomography (PAT) (Hoelen

et al

1998, Ku and Wang 2005, Ku

et al

2005,

Zhang

et al

2006, Song and Wang 2007, Song

et al

2008) that combines the advantages of optical

absorption contrast with ultrasonic spatial resolution has emerged as an excellent technique

for non-invasive imaging of biological tissues, offering high spatial resolution and high soft

tissue contrast.

In vivo

PA imaging of SLN in a rat model was also reported using methylene

blue (Song

et al

2008). However, methylene blue does not permit molecular imaging. Recently,

single-walled carbon nanotube (SWNT)-based contrast agents have shown promise for a

variety of imaging techniques. The prospect of SWNTs as a contrast agent for PA imaging was

also reported (Pramanik

et al

2009). In this study, we exploited the intrinsic optical absorbance

(Huges

et al

2007, Berciaud

et al

2007) of carbon nanotubes to develop them as contrast agents,

and used them to perform non-invasive imaging of SLN in a rat model

in vivo

2. Methods and Materials

2.1. Photoacoustic Imaging

A reflection-mode PA imaging system (Song and Wang 2007) was used to get the PA

spectroscopy of the SWNTs. A tunable Ti:sapphire laser (LT-2211A, LOTIS TII) pumped by

Q-switched Nd:YAG (LS-2137, LOTIS II) laser was the light source, providing <15 ns pulse

duration and a 10-Hz pulse repetition rate. A dark-field ring-shaped illumination was used

(Maslov

et al

2005). The light energy on the sample surface was controlled to conform to the

American National Standards Institute (ANSI) standard for maximum permissible exposure

(MPE) (ANSI 2000). A 3.5 MHz/5 MHz central frequency, spherically focused (4.95 cm/2.54

cm focus length, 1.91 cm diameter active area element, and 70%/72% bandwidth) ultrasonic

transducer (V380/V308, Panametrics-NDT) was used to acquire the generated PA signals. The

signal was then amplified by a low-noise amplifier (5072PR, Panametrics-NDT), and recorded

using a digital oscilloscope (TDS 5054, Tektronix) with a 50 mega-sampling rate. PA signal

fluctuations due to pulse-to-pulse energy variation were compensated by signals from a

photodiode (DET110, Thorlabs), which sampled the energy of each laser pulse.

A linear translation stage (XY-6060, Danaher Motion) was used for raster scanning to obtain

three-dimensional (3-D) PA data. A computer controlled the stage and synchronized it with

the data acquisition. To shorten the data acquisition time, a continuous scan was used without

signal averaging. An A-line (A-scan) was the PA signal obtained along the depth direction at

a single point. Multiple A-lines (acquired by a one-dimensional (1-D) scan) gave a two-

Pramanik et al.

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NIH-PA Author Manuscript

dimensional (2-D) B-scan. A 3-D image was acquired with a 2-D scan. A 1-D depth-resolved

image was obtained by multiplying the time axis of the initial A-scan (resolved in time along

the depth direction) by the speed of sound in soft tissue (~1500 m/s).

The scanning time depends on the laser pulse repetition rate (PRR), the scanning step size, and

the field of view (FOV). Typical values are a scanning step size for a 1-D scan = 0.1 mm, for

a 2-D scan = 0.2 mm, a laser PRR = 10 Hz, and a FOV = 20 mm × 20 mm. The acquisition

time = ~25 sec for a B-scan, and = ~42 min for a 3-D image. The transducer was located inside

a water container with an opening of 5 cm × 5 cm at the bottom, sealed with a thin, clear

membrane. The object was placed under the membrane, and ultrasonic gel was used for

coupling the sound.

2.2. SWNTs synthesis

Iron (Fe) was coated onto a silicon wafer using the diblock copolymer template method (Fu

et al

2004

). SWNTs were synthesized using the carbon vapor deposition (CVD) method with

Fe as the metal catalyst (Easy Tube 2000, First Nano). The wafer was placed into a 3 inch

quartz reaction chamber that was heated with Ar at 900°C, hydrogen gas was added for 2

minutes, and methane was fed into the reactor for 20 minutes to grow SWNTs over the Fe

catalyst. The SWNTs were further dispersed in 1 wt% Pluronic® F127 surfactant (1 g of

surfactant in 100 g of deionized water) at the appropriate concentration and sonicated

rigorously to obtain a homogeneous dispersion. To visualize the SWNT structure, transmission

electron microscopy (TEM) was performed (Tecnai12 BioTwinG2, FEI, Hillsboro, OR) at 80

kV on SWNT samples mounted on a 400 mesh copper grid with formvar coating. High

resolution TEM (hrTEM) (JEOL 2000 FX) was also performed at 200 kV for SWNT samples

mounted on a copper grid that was coated with amorphous carbon-holey film. In addition,

atomic force microscopy (AFM) was also performed (MFD-3D-BIO, Asylum Research, Santa

Barbara, CA). A Raman spectrum of the SWNTs was also taken at 633 nm (LabRam Aramis,

Horiba JvonYvon).

2.3. Animal and Drug Information

Guidelines on the care and the use of laboratory animals at Washington University in St. Louis

were followed for all animal experiments. Adult Sprague Dawley rats with various body

weights (250 – 350 g) were used for the experiments. Initial anesthetization of the rat was done

using a mixture of ketamine (85 mg/kg) and xylazine (15 mg/kg). The hair on the region of

interest of the rat was gently removed before imaging, using a commercial hair-removal lotion.

Intradermal injection of 0.075 ml of 0.5 mg/ml SWNTs was performed on a left/right forepaw

pad, depending on which side was imaged. PA images were acquired after the administration

of SWNTs. During the image acquisition, anesthesia was maintained using vaporized

isoflurane (1 L/min oxygen and 0.75% isoflurane, Euthanex Corp.), and a pulse oximeter

(NONIN Medical INC., 8600V) was used to monitor the vitals. If needed, 8 ml of 0.9% saline

was administered to the rat for hydration. After image acquisition, the animal was euthanized

by pentobarbital overdose.

3. Results and Discussions

3.1. Characterization of SWNTs

Figure 1 shows the TEM, hrTEM, and Raman spectroscopy of the synthesized SWNTs used

in this study. These images were used to characterize the SWNTs. Figures 1(a) and 1(b) show

low-resolution bright field TEM and the hrTEM images of the SWNTs, respectively. These

two figures confirm the growth of SWNTs with an approximate diameter of 2 nm. Figure 1(c)

shows an AFM image, which confirms that the SWNT diameters were consistent with the

diameters observed in the hrTEM image. Figure 1(d) shows the Raman spectrum of the SWNTs

Pramanik et al.

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NIH-PA Author Manuscript

at 633 nm. The Raman spectrum shows the G band at 1592 cm

−

and the D band at 1315

−

. The G band is a peak specific to graphene, while the D band indicates defects on the

graphene sheet. The low D band to G band ratio signifies few defects in the SWNT sample.

The radial breathing modes, which occur in the presence of small diameter tubes of a single

graphene layer, can be viewed within the inset, and further confirm that these samples were

SWNTs (Dresselhaus

et al

2005).

3.2. Photoacoustic spectroscopy of SWNTs

Blood is a dominant optical absorber in the human body and produces strong PA signals.

Therefore, the PA signal from SWNTs was compared with that from blood to show that,

SWNTs are capable of generating PA signals comparable to or stronger than that of a known

absorber in the body in the near infrared (NIR) wavelength window. Figure 2(a) shows the PA

signals obtained from a tygon tube (I.D. 250

m, O.D. 500

m) filled with SWNTs (0.25 mg/

ml) and rat blood. The laser was tuned to 764 nm wavelength. At this excitation wavelength,

the peak-to-peak PA signal amplitude obtained from SWNTs was ~600 mV, compared to a

~170 mV peak-to-peak PA signal amplitude from blood alone. Figure 2(b) shows the PA

spectrum (peak-to-peak PA signal amplitude versus excitation light wavelength) of the SWNTs

(in black) for an excitation wavelength range of 740–820 nm. The PA spectrum of rat blood

(in red) is also shown in the same figure. It is evident that the PA signal obtained from SWNTs

is much stronger than that of blood over the entire wavelength range. Therefore, one can choose

a specific light wavelength for imaging within a broad range. Figure 2(c) plots the ratio of the

peak-to-peak PA signal amplitude of SWNTs to that of blood between 740 and 820 nm. The

PA signal from the tygon tube filled with SWNTs is more than four times stronger than that

from blood at 750 nm. Over the entire 740–820 nm window, the PA signal from SWNTs is

more than two times stronger than that from blood. Due to the weak blood absorption, the NIR

window is well known for providing deep tissue PA imaging at the expense of blood contrast.

The strong PA signal from SWNTs in the NIR region implies that they could be used as contrast

agents to boost the signal strength for PA imaging in this region.

3.3. Deep tissue imaging

The sensitivity of the PA imaging system was determined from chicken tissue phantom

experiments, used to mimic human breast tissue (

Song

et al

2008

). The light source was tuned

to 793 nm wavelength. A tube (Silastic® laboratory tubing, Dow Corning Corp.) with 1.47

mm I.D. was placed between two layers of chicken breast tissue. The tube was filled with 1

mg/ml SWNTs. The thickness of the tissue layer on top of the tube was ~20 mm. Figure 3(a)

shows the maximum amplitude projection (MAP) (Zhang

et al

2006) image of the tube. The

tube is clearly seen in the image, with a high contrast-to-noise ratio (CNR) of 25. Figure 3(b)

shows the B-scan PA image (along the dotted line in figure 3(a)). The bright spot in the B-scan

represents the PA signal generated from the tube filled with SWNTs. Figure 3(c) shows the A-

line PA signal (along the dotted line in figure 3(b)), clearly showing the strong PA signal from

the tube filled with SWNTs compared to the weak signal from the surrounding chicken breast

tissue. These results prove that use of SWNTs as a contrast agent enables tissue imaging more

than 20 mm deep. For human studies, the depth of imaging is very important, as the mean depth

of SLNs is 12±5 mm (distance from the skin surface to the top surface of SLN) (

Margenthaler

2007). Therefore, the ability to image more than 20 mm deep with high CNR and high

resolution is a key for mapping SLN non-invasively.

3.4. Sentinel lymph node imaging non-invasively in a rat in vivo

Adult Sprague Dawley rats with various body weights (250 – 350 g) were used for the

experiments. The hair on the axillary region of the rat was removed and a PA image was taken.

This image was a control image for comparison with future images. An intradermal injection

Pramanik et al.

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NIH-PA Author Manuscript

of 0.075 ml of 0.5 mg/ml SWNTs was performed on the forepaw pad. Four PA images were

acquired at intervals of 25–30 minutes after the SWNTs injection. For all the PA images, the

following parameters were used: FOV = 25 mm × 30 mm, step size along the X direction =

0.2 mm, step size along the Y direction = 0.4 mm, total scan time = ~23 minutes. Please note

that no signal averaging was done for any of these images. The images shown here are cropped

to a FOV of 16 mm × 24 mm, since the outside region was not of interest.

Figure 4(a) shows a representative digital photograph of a rat taken prior to image acquisition,

and figure 4(b) shows the shaved axillary surface where the PA imaging was performed. Before

the SWNTs injection, a PA control image was obtained, which is shown in the form of a MAP

(Zhang

et al

2006) in figure 4(c). The vasculature near an axial node (one blood vessel is marked

as BV) was clearly imaged, with a high CNR of ~79 and good resolution of ~500

m. Note

that no lymph nodes are visible in the control image, since there is no intrinsic optical absorption

in lymph nodes to produce any PA signal to image. Figure 4(d) shows the PA image (MAP)

of the same area immediately after the SWNTs were injected, approximately 30 min after the

control image was taken. Figures 4(e), 4(f), and 4(g) are the post-injection PA images (MAP)

of the same area 30 min, 55 min, and 85 min after the SWNTs injection. The SLN appears at

the left lower quadrant, marked as SLN in figure 4(f), and is clearly visible in all the post-

injection PA images (Figures 4(d), 4(e), 4(f), and 4(g)). The images show high CNRs (74 in

figure 4(d), 67 in figure 4(e), 84 in figure 4(f), and 89 in figure 4(g)). The contrast of the SLN

to the surrounding blood vessel was up to 1.8 (ratio of the peak-to-peak PA signal amplitude

obtained from SLN and BV) after SWNTs injection. The signal amplitude of the surrounding

blood vessels was also increased by up to ~124% compared to that in the control image (94%

in figure 4(d), 110% in figure 4(e), 124% in figure 4(f), and 105% in figure 4(g)), although the

CNR remained almost the same. This increase in signal amplitude suggests that the SWNTs

have traveled into the blood stream and the nearby tissues boosting the blood vessel signal as

well as the background signal. Figure 4(h) is a digital photograph of the same rat with the skin

removed after the completion of the PA imaging. Figure 4(i) is a digital photograph of the SLN

removed from the rat (arrow in figure 4(i)). The photograph shows the SLN size to be 2–3 mm,

matching the size obtained from PA images (Figures 4(d) to 4(g)).

Figure 5(a) shows the uptake kinetics of the SWNTs. An increased PA signal was observed in

both blood vessels and SLN during the first 30 minutes post injection, with the SLN showing

a greater increase than blood vessels at all time points. Subsequently, the PA signal in the blood

reached a plateau while the PA signal in the SLN kept increasing. These results further

corroborate the imaging data presented in figure 4, where increase in signal intensities were

observed post injection from both SLN and BV, but the intensity from SLN was stronger

compared to the nearby blood vessels. The stronger signal is due to the strong light absorption

of SWNTs compared to blood, and to the greater accumulation of SWNTs in the lymph node

than in the surrounding blood vessels. Figures 5(b) and 5(c) show the B-scan PA images

corresponding to the dotted lines marked in figures 4(c) and 4(f), respectively. The bright spot

in figure 5(c) represents the strong PA signal from the SLN, which is not present in the pre-

injection B-scan (Figure 5(b)). Figures 5(d) and 5(e) show the A-line PA signals corresponding

to the dotted lines in figures 5(b) and 5(c), respectively. The strong PA signal obtained from

the SLN is clearly seen in figure 5(e), whereas there is no such signal in the control image

(Figure 5(d)). We used a 0.5 mg/ml concentration of SWNTs for our

in vivo

study (the average

molecular weight of SWNTs is ~10

Da or g/mol; 0.5 mg/ml = 0.5 mg/ml/10

g/ml = 500 nM).

However, that choice does not limit the use of SWNTs at other lower concentrations (De La

Zerda A

et al

2008). Our system is capable of detecting SWNTs on the order of nM

concentration (Pramanik

et al

2009).

Our results show that non-invasive

in vivo

PA imaging for SLN identification with the use of

SWNTs as a contrast agent is highly feasible in a small animal model. Moreover, the PA

Pramanik et al.

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NIH-PA Author Manuscript

imaging technique used here to image rat SLN can easily be translated to humans. Although,

SLN mapping using different contrast agents, e.g., carbon nanotubes, gold nanocages, gold

nanorods, and methylene blue dye, has been done successfully, we believe SWNTs have many

advantages over the other materials. The broad absorption spectrum of SWNTs will provide

us a wide range of light wavelengths for imaging. Most of the other contrast agents have narrow

absorption spectra, restricting their use to a particular wavelength. SWNTs also show good

efficacy as contrast agents for thermoacoustic imaging (Pramanik

et al

2009). In the future,

thermoacoustic tomography can also be combined with PA imaging system for SLN mapping.

Targeted SWNTs could also be used for more specific identification. Our previous PA studies

demonstrated that SWNTs produce a PA signal at concentrations as low as 0.1 mg/ml

(Pramanik

et al.

2009). Using a 0.5 mg/ml concentration of SWNTs, we have obtained very

high CNR sentinel lymph node images (CNR = ~90), thus it can be inferred that we could

detect SWNTs at concentrations as low as 0.01 mg/ml. Assuming the SWNTs have an average

diameter of 2 nm and average length of 1

m, there should be ~ 10

carbon atoms in one SWNT,

as derived by Yamamoto

et al

2005, and a SWNT has a molecular weight of ~1.2 × 10

g/mol.

Using these values, SWNTs should act as contrast agents in human blood at concentrations as

low as 8 nM (0.1/1.2 × 10

). SWNTs can also be functionalized in order to target specific

tissues and to increase the SWNTs blood circulation time. This would prevent the SWNTs

from being diluted in the human bloodstream for periods long enough to perform PA imaging.

Currently, this imaging system is limited by its slow scanning speed. Employing a higher pulse-

repetition-frequency laser and an ultrasound array system could accelerate acquisition,

potentially allowing real-time PA imaging (Yin

et al

2004, Zeng

et al

2004, Yang

et al

2005). The

in vivo

biocompatibility of SWNTs needs to be thoroughly examined before its

translation for clinical use. Nevertheless, since SLN identification by PA imaging is totally

non-invasive and safe, it shows potential future clinical applications without the limitations of

current invasive and minimally-invasive techniques.

4. Conclusions

We have shown that the PA signal from SWNTs is stronger than that of blood over the NIR

wavelength window (740–820 nm) and demonstrated a non-invasive SWNTs-enhanced PA

identification of SLN in a rat model

in vivo

with a high contrast-to-noise ratio (CNR = 89) and

good resolution (~500

m). We have also shown the possibility of deep tissue imaging (20 mm

deep) using SWNTs as contrast agent. Our results suggest that this technology could be a useful

pre-clinical and possibly clinical tool to identify SLNs non-invasively

in vivo

. In the future,

the identification rate of node-negative breast cancer could be improved by functionalization

of the SWNTs with targeting groups.

Acknowledgments

This work was supported by National Institutes of Health grants (R01 EB000712, R01 NS46214 (Bioengineering

Research Partnerships), R01 EB008085, and U54 CA136398 (Network for Translational Research) - LVW) and the

Office of the Vice President of Research at Stony Brook University, Carol M. Baldwin fund (SB). L.W. has a financial

interest in Endra, Inc., which, however, did not support this work. The authors would like to thank Dr. Oleg Gang and

Dr. Huming Xiong at the Center for Functional Nanomaterials, Brookhaven National Laboratory for access to the

AFM, Mr. Tom Salagaj and Mr. Christopher Jensen at FirstNano/CVD Equipment Corporation for access to their

CVD facilities and Dr. Eunah Lee at Horiba JvonYvon, Edison, NJ for the Raman Spectroscopy measurements.

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and noninvasive

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Pramanik et al.

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Figure 1.

SWNT characterization. (a) TEM and (b) hrTEM image of the SWNTs produced by the Fe

catalyst. (c) AFM image of the SWNTs produced by the Fe catalyst, confirming the nanotube

diameter of 2 nm. (d) A Raman spectrum of the SWNT obtained at 633 nm. The G band is at

1592 cm

−

, while the D band is found at 1315 cm

−

. The radial breathing modes occur between

100 and 350 cm

−

(see inset).

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Figure 2.

(a) PA signals generated from a tygon tube (I.D. 250

m, O.D. 500

m) filled with SWNTs

(0.25 mg/ml) and rat blood. The excitation optical wavelength is 764 nm. (b) PA spectra of

SWNTs and blood over a 740–820 nm range of NIR wavelengths. (c) Ratio of the peak-to-

peak PA signal amplitudes generated from SWNTs to those generated from blood.

Pramanik et al.

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Figure 3.

(a) Maximum Amplitude Projection (MAP) image of a tube (Silastic® laboratory tubing, Dow

Corning Corp., with 1.47 mm I.D.) filled with SWNTs (1 mg/ml) placed inside chicken breast

tissue. The tube was placed 20 mm below the top surface of the tissue. The PA image clearly

shows the tube, with a CNR of 25. (b) PA B-scan image along the dotted line in figure 3(a),

with the bright spot showing the PA signal originating from the tube filled with SWNTs. (c)

Photoacoustic A-line along the dotted line in figure 3(b).

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Figure 4.

Non-invasive

in vivo

PA images (MAP) of the SLN in a rat. For all PA images, the laser was

tuned to 793 nm wavelength. (a) Photograph of the rat. (b) Photograph of the rat after the hair

was removed from the scanning region before taking the PA images. The scanning region is

marked with a black dotted square. (c) Control PA image acquired before SWNTs injection.

Bright parts represent optical absorption, here, from blood vessels (BV). (d) PA image (MAP)

acquired immediately after the SWNTs injection. (e) 30 min post-injection PA image. (f) 55

min post-injection PA image. (g) 85 min post-injection PA image. Blood vessel (BV) and

sentinel lymph node (SLN) are marked with arrows, and the SLN is visible in all images except

the control image. (h) Photograph of the rat with the skin removed after PA imaging. (i) The

excised lymph node. For Figures 4(c) to 4(g): FOV = 25 mm × 30 mm, step size along the X

direction = 0.2 mm, step size along the Y direction = 0.4 mm, total scan time = ~23 minutes.

No signal averaging was used. Only a FOV of 16 mm × 24 mm is shown.

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Figure 5.

(a) Peak-to-peak PA signal amplitude obtained from SLN and blood vessel (BV) over time.

The SWNTs were injected 30 min after the control scan. (b) PA B-scan image corresponding

to the dotted line in figure 4(c), the control image. (c) PA B-scan image corresponding to the

dotted line in figure 4(f), image showing the SLN. The bright spot represents the PA signal

generated from the SLN. (d) A-line PA signal corresponding to the dotted line in (b). (e) A-

line PA signal corresponding to the dotted line in (c), showing the strong PA signal generated

from the SLN.

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