Due to the wide use of animals for human disease studies, small animal whole-body imaging plays an increasingly important role in biomedical research. Currently, none of the existing imaging modalities can provide both anatomical and glucose metabolic information, leading to higher costs of building dual-modality systems. Even with image coregistration, the spatial resolution of the metabolic imaging modality is not improved. We present a ring-shaped confocal photoacoustic computed tomography (RC-PACT) system that can provide both assessments in a single modality. Utilizing the novel design of confocal full-ring light delivery and ultrasound transducer array detection, RC-PACT provides full-view cross-sectional imaging with high spatial resolution. Scanning along the orthogonal direction provides three-dimensional imaging. While the mouse anatomy was imaged with endogenous hemoglobin contrast, the glucose metabolism was imaged with a near-infrared dye-labeled 2-deoxyglucose. Through mouse tumor models, we demonstrate that RC-PACT may be a paradigm shifting imaging method for preclinical research

Chatni, Muhammad

Maslov, Konstantin

Wang, Lihong V.

Xia, Jun

Caltech Authors - Main

PROCEEDINGS OF SPIE
SPIEDigitalLibrary.org/conference-proceedings-of-spie
Anatomical and metabolic small-
animal whole-body imaging using
ring-shaped confocal photoacoustic
computed tomography
Jun  Xia, Muhammad  Chatni, Konstantin  Maslov, Lihong
V. Wang
Jun  Xia, Muhammad  Chatni, Konstantin  Maslov, Lihong V. Wang,
"Anatomical and metabolic small-animal whole-body imaging using ring-
shaped confocal photoacoustic computed tomography," Proc. SPIE 8581,
Photons Plus Ultrasound: Imaging and Sensing 2013, 85810K (4 March
2013); doi: 10.1117/12.2004937
Event: SPIE BiOS, 2013, San Francisco, California, United States
Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 9/5/2018  Terms of Use: https://www.spiedigitallibrary.org/terms-of-use
  
Anatomical and metabolic small-animal whole-body imaging using 
ring-shaped confocal photoacoustic computed tomography 
Jun Xia, Muhammad Chatni, Konstantin Maslov, and Lihong V. Wang* 
Department of Biomedical Engineering, One Brookings Drive, Washington University in St. Louis, 
St. Louis, MO, USA 63130 
*Correspondence should be addressed to lhwang@biomed.wustl.edu 
ABSTRACT  
Due to the wide use of animals for human disease studies, small animal whole-body imaging plays an increasingly 
important role in biomedical research. Currently, none of the existing imaging modalities can provide both anatomical 
and glucose metabolic information, leading to higher costs of building dual-modality systems. Even with image co-
registration, the spatial resolution of the metabolic imaging modality is not improved. We present a ring-shaped confocal 
photoacoustic computed tomography (RC-PACT) system that can provide both assessments in a single modality. 
Utilizing the novel design of confocal full-ring light delivery and ultrasound transducer array detection, RC-PACT 
provides full-view cross-sectional imaging with high spatial resolution. Scanning along the orthogonal direction provides 
three-dimensional imaging. While the mouse anatomy was imaged with endogenous hemoglobin contrast, the glucose 
metabolism was imaged with a near-infrared dye-labeled 2-deoxyglucose. Through mouse tumor models, we 
demonstrate that RC-PACT may be a paradigm shifting imaging method for preclinical research. 
Keywords: Photoacoustic computed tomography, small-animal whole-body imaging, metabolic imaging 
 
1. INTRODUCTION  
The importance of imaging tumor metabolism establishes itself from the premise that anatomical changes alone are not 
an accurate metric for diagnosis, prognosis, and therapy. In particular, tumor metabolic imaging provides a spatial and 
temporal map of therapeutic response and guides treatment planning and selection. Therefore, simultaneous metabolic 
and anatomical imaging is very desirable, and necessary not only in clinical settings, but also in preclinical cancer 
models to improve our understanding of cancer, metastasis, and drug efficacy in vivo [1, 2].  
Currently, X-ray computed tomography (CT) [3] and magnetic resonance imaging (MRI) [4] provide high-resolution 
anatomical images, but they cannot provide sufficient metabolic contrast without combining with metabolic imaging 
modalities. Despite the high cost of dual-modality imaging systems and the need for image registration, metabolic 
imaging systems have their own limitations. For instance, positron emission tomography (PET) is very expensive, and 
the radioisotopes are difficult to manufacture and have short half-lives. In addition, the accumulated radiation dosage of 
the combined PET/CT may be carcinogenic and will confound results in oncology [5, 6].  
Here, we present a ring-shaped confocal photoacoustic computed tomography (RC-PACT) scanner that can image both 
anatomy and tumor glucose metabolism at high spatial resolution in a single imaging modality [7, 8]. In RC-PACT, 
anatomical images were produced by endogenous hemoglobin contrast, and tumor glucose metabolism was imaged and 
quantified using spectral separation of the absorption of IRDye800-2DG, a near-infrared fluorophore-labeled glucose 
analog, from that of hemoglobin [9]. With the unique design of confocal free-space full-ring light illumination and full-
ring ultrasound transducer array detection, our RC-PACT system generates in vivo cross-sectional images with 
simultaneous anatomical and metabolic assessments at high spatial resolution [10].  
2. SYSTEM DESIGN 
Figure 1 shows the schematic of the RC-PACT system [11]. A tunable Ti-Sapphire laser with 12 ns pulse duration and 
10 Hz pulse repetition rate was used as the irradiation source. The laser beam passed through a conical lens to form a 
ring-shaped light, which was then focused using an optical condenser to project a thin light band around the object. The 
light incident area was aligned to be slightly above the acoustic focal plane to minimize the detection of strong surface 
Photons Plus Ultrasound: Imaging and Sensing 2013, edited by Alexander A. Oraevsky, Lihong V. Wang,
Proc. of SPIE Vol. 8581, 85810K · © 2013 SPIE · CCC code: 1605-7422/13/$18 · doi: 10.1117/12.2004937
Proc. of SPIE Vol. 8581  85810K-1
Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 9/5/2018
Terms of Use: https://www.spiedigitallibrary.org/terms-of-use
Conical lens
Anesthesia gas tube
c - - Optical condenser
! Water
Transducer array
Animal
Heater
Flexible
membrane
agnetic base
512 preamps
8:1 multiplexers
64- channel
amplifier& DAQ
Ti- sapphire laser
1
Computer
,,,..,
1
 
 
signals. The photoacoustic signals were detected by a 512-element full-ring transducer array with 5 MHz central 
frequency (80% bandwidth) and 50 mm ring diameter. Each element in the array was mechanically shaped into an arc to 
produce an axial focal depth of 19 mm within the imaging plane. The combined foci of all elements generated a central 
imaging region of 20 mm diameter and 1 mm thickness [11, 12]. Since the light incidence was oblique, the light formed 
a weak focus inside the animal body. This focal region overlapped with the acoustic focal plane to improve the 
efficiency of detecting photoacoustic signals generated in deep tissues [7]. 
 
 
 
 
Figure 1. Experimental setup of the full-ring confocal whole-body photoacoustic computed tomography system. 
 
3. SYSTEM CALIBRATION 
In spectral experiments, the beam profile of the laser varied at different wavelengths, which led to changes in the fluence 
distribution, adding complexities on spectral separation. This problem can be solved either by aligning the optics after 
each wavelength tuning, which requires a tremendous amount of time in three-dimensional scanning, or more efficiently, 
by post-experimental compensation. In this research, we used a phantom calibration method to compensate for both the 
laser power and the light band uniformity of different wavelengths. The phantom was made from a gelatin-water 
suspension mixed with 0.1% black ink. The suspension was put in a cylindrical container, whose inner diameter was 
similar to the mouse cross-sectional diameter (20 mm), and was then cooled in the fridge until gelled. The gelled 
phantom was then mounted on the RC-PACT scanner and imaged using the same scheme as the animal experiment. 
Cross-sectional photoacoustic images of the phantom were reconstructed at each wavelength, and the ring-shaped light 
variation was obtained based on the surface signal of the phantom image. The matrix of light variation was then used as 
a weighting function to calibrate the spectral in vivo images. The calibration results are demonstrated in Figure 2. The 
compensated images have similar signal intensity at different wavelengths and a more uniform signal distribution around 
the illumination ring.  
Proc. of SPIE Vol. 8581  85810K-2
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5 mm
(a) (b) (c)
(d) (e) (f)
(g) (h) (i)
776 nm 796 nm 820 nm
PA
 a
m
pl
itu
de
 (
a.
u.
)
Max
Min  
Figure 2. Image compensation for the wavelength-dependent laser power and ring-shaped-light distribution. Top row (a-c), un-
compensated images. Middle row (d-f), phantom images. Bottom row (g-i), compensated images. Left column: images acquired at 
776 nm. Middle column: images acquired at 796 nm. Right column: images acquired at 820 nm. 
 
4. IN VIVO ANATOMICAL IMAGING 
To demonstrate the in vivo anatomical imaging capability of our RC-PACT system, we imaged healthy athymic (nude) 
mice. Figure 3 shows serial cross-sectional images of 5-6-week-old healthy athymic mice in axial views. The brain 
cortex, heart, liver, spleen, and kidneys are clearly visible. Additionally, detailed vascular structures within these organs 
are visible, demonstrating that the system can be used for whole-body angiographic imaging without injecting 
exogenous contrasts. The spinal cord, stomach, and gastrointestinal tracts are imaged due to the surrounding 
microvasculature. Major blood vessels, such as the vena cava, are also clearly visible. Using exogenous optical contrast 
(e.g., near-infrared dyes), the system can also image organs with little blood. The bladder image was acquired 30 
minutes after tail vein administration of IRDye800-2DG. The urinary bladder showed strong contrast as it was filled 
with the dye excreted by the kidneys. 
Proc. of SPIE Vol. 8581  85810K-3
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l 1
 
 
Liver
Heart
Brain
Kidneys
Intestine
Bladder
 
Figure 3. RC-PACT images of athymic mice acquired noninvasively at various anatomical locations: 
  
5. IN VIVO TUMOR GLUCOSE METABOLISM IMAGING 
To showcase the capability of our RC-PACT system to image the glucose metabolism of deeper tumors, we imaged two 
mice with orthotopically implanted 786-O tumor cells in their left kidneys.  
Following the control experiment, 200 µL of (0.4 mM) IRDye800-2DG was administered, and the mice were imaged 
after 24 hours. This 24-hour time window is sufficient for excess unbound IRDye800-2DG to be excreted by the kidneys 
and bladder. This also increases the likelihood that the imaged IRDye800-2DG reflects tumor glucose metabolism rather 
than the physical entrapment of excess dye molecules inside the tumors. Figures 4(a) show representative anatomical 
cross-sections of the two 786-O tumor mice. The HbT concentrations were normalized by the maximum HbT 
concentration (observed in the vena cava), scaled to 2.3 mM [Figures 4(b)], and then the absolute IRDye800-2DG 
concentrations in the anatomical cross-section were estimated. Images of tumor glucose metabolism [figures 4(c)] were 
overlaid on the gray-scale PACT images acquired at 776 nm. In Figures 4(c), the mean IRDye800-2DG concentration in 
healthy kidneys were used as the threshold to account for non-specific IRDye800-2DG uptake due to kidneys being the 
excretory route. The signal-to-noise ratio for imaging IRDye800-2DG in the most metabolically active site in the tumor 
was 39.3 dB for mouse 1 and 38.3 dB for mouse 2. The tumor-to-normal tissue contrast for IRDye800-2DG uptake was 
approximately 3.3 at the most metabolically active site in the tumor. The same image reconstruction and spectral 
separation methods were also applied on the control RC-PACT images, and no significant IRDye800-2DG uptake was 
observed. 
 
Proc. of SPIE Vol. 8581  85810K-4
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Awl
PA am 1"rtu& (á.u.
(c)
[Hal ("RAI)
10 16 20 26 30
-2-r- -1 a 9 fir- 3
PA amplitude (a.u.)
AMIN=
12 lE3 "IS
PG11 (11M)
iF 04 1.2 14 2.1. 3.0 d.
[i113u J
 
 
 
 
 
Figure 4. In vivo RC-PACT images of orthotopically implanted 786-O kidney tumors. Top row: mouse 1. Bottom row: mouse 2. (a) 
Anatomical images acquired at 776 nm. (b) HbT images overlaid on the anatomical images. (c) IRDye800-2DG images overlaid on 
the anatomical images. CK, cancerous kidney; HK, healthy kidney; HM, hypermetabolic. 
 
  
6. DISCUSSION AND CONCLUSION 
Anatomical and metabolic imaging is of great importance due to its wide application in tumor biology, drug discovery, 
and early-stage cancer screening in both clinical and pre-clinical settings. In this report, we have demonstrated that RC-
PACT provided tumor glucose metabolism and anatomy images with ultrasound-defined spatial resolution in a single 
imaging modality. While PET-CT and PET-MRI have been well established in the field, RC-PACT is a promising and 
less expensive alternative with the advantages of non-ionizing laser radiation, non-radioactive optical contrast, and 
endogenous hemoglobin contrast. Additionally, image co-registration of anatomical and metabolic assessments is 
automatic. Besides imaging tumor glucose metabolism, RC-PACT can also benefit from near-infrared fluorescent dyes 
[8] and proteins [13] for deep molecular imaging along with detailed, high resolution anatomical structures. The 
possibility of multiplexing functional parameters in combination with anatomical imaging uniquely positions RC-PACT 
to have a broad impact in preclinical medical research and life sciences. Due to the fixed ring diameter of the current 
system, we only demonstrated 1 cm penetration depth; however, photoacoustic imaging at depth of 8.4 cm has been 
Proc. of SPIE Vol. 8581  85810K-5
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reported [14]. Taking advantage of the full-ring light illumination, which doubles the imaging depth (~16.8 cm), RC-
PACT can potentially be scaled to image larger animals or even humans. 
7. ACKNOWLEDGEMENTS 
The authors appreciate Ms. Seema Dahlheimer’s close reading of the manuscript. This work was sponsored in part by 
National Institutes of Health grants R01 EB000712, R01 EB008085, R01 CA134539, U54 CA136398, R01 EB010049, 
R01 CA157277, R01 CA159959, and 5P60 DK02057933. L.W. has financial interests in Microphotoacoustics, Inc. and 
Endra, Inc., which, however, did not support this work. 
REFERENCES 
[1] M. Baker, “Whole-animal imaging: The whole picture,” Nature, 463(7283), 977-980 (2010). 
[2] R. A. de Kemp, F. H. Epstein, C. Catana et al., “Small-Animal Molecular Imaging Methods,” Journal of 
Nuclear Medicine, 51(Supplement 1), 18S-32S (2010). 
[3] D. W. Holdsworth, and M. M. Thornton, “Micro-CT in small animal and specimen imaging,” Trends in 
Biotechnology, 20(8), S34-S39 (2002). 
[4] H. Benveniste, and S. Blackband, “MR microscopy and high resolution small animal MRI: applications in 
neuroscience research,” Progress in Neurobiology, 67(5), 393-420 (2002). 
[5] G. Brix, U. Lechel, G. Glatting et al., “Radiation Exposure of Patients Undergoing Whole-Body Dual-Modality 
18F-FDG PET/CT Examinations,” Journal of Nuclear Medicine, 46(4), 608-613 (2005). 
[6] R. Fazel, H. M. Krumholz, Y. Wang et al., “Exposure to Low-Dose Ionizing Radiation from Medical Imaging 
Procedures,” New England Journal of Medicine, 361(9), 849-857 (2009). 
[7] J. Xia, M. Chatni, K. Maslov et al., “Whole-body ring-shaped confocal photoacoustic computed tomography of 
small animals in vivo,” Journal of Biomedical Optics, 17(5), 050506 (2012). 
[8] J. Yao, J. Xia, K. I. Maslov et al., “Noninvasive photoacoustic computed tomography of mouse brain 
metabolism in vivo,” NeuroImage, 64, 257-266 (2013). 
[9] J. L. Kovar, W. Volcheck, E. Sevick-Muraca et al., “Characterization and performance of a near-infrared 2-
deoxyglucose optical imaging agent for mouse cancer models,” Analytical Biochemistry, 384(2), 254-262 
(2009). 
[10] M. R. Chatni, J. Xia, R. Sohn et al., “Tumor glucose metabolism imaged in vivo in small animals with whole-
body photoacoustic computed tomography,” Journal of Biomedical Optics, 17(7), 076012 (2012). 
[11] J. Xia, Z. Guo, K. Maslov et al., “Three-dimensional photoacoustic tomography based on the focal-line 
concept,” Journal of Biomedical Optics, 16(9), 090505 (2011). 
[12] J. Gamelin, A. Maurudis, A. Aguirre et al., “A real-time photoacoustic tomography system for small animals,” 
Opt. Express, 17(13), 10489-10498 (2009). 
[13] G. S. Filonov, A. Krumholz, J. Xia et al., “Deep-Tissue Photoacoustic Tomography of a Genetically Encoded 
Near-Infrared Fluorescent Probe,” Angewandte Chemie International Edition, 51(6), 1448-1451 (2012). 
[14] H. Ke, T. N. Erpelding, L. Jankovic et al., “Performance characterization of an integrated ultrasound, 
photoacoustic, and thermoacoustic imaging system,” Journal of Biomedical Optics, 17(5), 056010-1 (2012). 
 
 
 
Proc. of SPIE Vol. 8581  85810K-6
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Anatomical and metabolic small-animal whole-body imaging using ring-shaped confocal photoacoustic computed tomography

PROCEEDINGS OF SPIE
SPIEDigitalLibrary.org/conference-proceedings-of-spie
Anatomical and metabolic small-
animal whole-body imaging using
ring-shaped confocal photoacoustic
computed tomography
Jun  Xia, Muhammad  Chatni, Konstantin  Maslov, Lihong
V. Wang
Jun  Xia, Muhammad  Chatni, Konstantin  Maslov, Lihong V. Wang,
"Anatomical and metabolic small-animal whole-body imaging using ring-
shaped confocal photoacoustic computed tomography," Proc. SPIE 8581,
Photons Plus Ultrasound: Imaging and Sensing 2013, 85810K (4 March
2013); doi: 10.1117/12.2004937
Event: SPIE BiOS, 2013, San Francisco, California, United States
Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 9/5/2018  Terms of Use: https://www.spiedigitallibrary.org/terms-of-use
 
 
Anatomical and metabolic small-animal whole-body imaging using 
ring-shaped confocal photoacoustic computed tomography 
Jun Xia, Muhammad Chatni, Konstantin Maslov, and Lihong V. Wang* 
Department of Biomedical Engineering, One Brookings Drive, Washington University in St. Louis, 
St. Louis, MO, USA 63130 
*Correspondence should be addressed to lhwang@biomed.wustl.edu 
ABSTRACT  
Due to the wide use of animals for human disease studies, small animal whole-body imaging plays an increasingly 
important role in biomedical research. Currently, none of the existing imaging modalities can provide both anatomical 
and glucose metabolic information, leading to higher costs of building dual-modality systems. Even with image co-
registration, the spatial resolution of the metabolic imaging modality is not improved. We present a ring-shaped confocal 
photoacoustic computed tomography (RC-PACT) system that can provide both assessments in a single modality. 
Utilizing the novel design of confocal full-ring light delivery and ultrasound transducer array detection, RC-PACT 
provides full-view cross-sectional imaging with high spatial resolution. Scanning along the orthogonal direction provides 
three-dimensional imaging. While the mouse anatomy was imaged with endogenous hemoglobin contrast, the glucose 
metabolism was imaged with a near-infrared dye-labeled 2-deoxyglucose. Through mouse tumor models, we 
demonstrate that RC-PACT may be a paradigm shifting imaging method for preclinical research. 
Keywords: Photoacoustic computed tomography, small-animal whole-body imaging, metabolic imaging 
 
1. INTRODUCTION  
The importance of imaging tumor metabolism establishes itself from the premise that anatomical changes alone are not 
an accurate metric for diagnosis, prognosis, and therapy. In particular, tumor metabolic imaging provides a spatial and 
temporal map of therapeutic response and guides treatment planning and selection. Therefore, simultaneous metabolic 
and anatomical imaging is very desirable, and necessary not only in clinical settings, but also in preclinical cancer 
models to improve our understanding of cancer, metastasis, and drug efficacy in vivo [1, 2].  
Currently, X-ray computed tomography (CT) [3] and magnetic resonance imaging (MRI) [4] provide high-resolution 
anatomical images, but they cannot provide sufficient metabolic contrast without combining with metabolic imaging 
modalities. Despite the high cost of dual-modality imaging systems and the need for image registration, metabolic 
imaging systems have their own limitations. For instance, positron emission tomography (PET) is very expensive, and 
the radioisotopes are difficult to manufacture and have short half-lives. In addition, the accumulated radiation dosage of 
the combined PET/CT may be carcinogenic and will confound results in oncology [5, 6].  
Here, we present a ring-shaped confocal photoacoustic computed tomography (RC-PACT) scanner that can image both 
anatomy and tumor glucose metabolism at high spatial resolution in a single imaging modality [7, 8]. In RC-PACT, 
anatomical images were produced by endogenous hemoglobin contrast, and tumor glucose metabolism was imaged and 
quantified using spectral separation of the absorption of IRDye800-2DG, a near-infrared fluorophore-labeled glucose 
analog, from that of hemoglobin [9]. With the unique design of confocal free-space full-ring light illumination and full-
ring ultrasound transducer array detection, our RC-PACT system generates in vivo cross-sectional images with 
simultaneous anatomical and metabolic assessments at high spatial resolution [10].  
2. SYSTEM DESIGN 
Figure 1 shows the schematic of the RC-PACT system [11]. A tunable Ti-Sapphire laser with 12 ns pulse duration and 
10 Hz pulse repetition rate was used as the irradiation source. The laser beam passed through a conical lens to form a 
ring-shaped light, which was then focused using an optical condenser to project a thin light band around the object. The 
light incident area was aligned to be slightly above the acoustic focal plane to minimize the detection of strong surface 
Photons Plus Ultrasound: Imaging and Sensing 2013, edited by Alexander A. Oraevsky, Lihong V. Wang,
Proc. of SPIE Vol. 8581, 85810K · © 2013 SPIE · CCC code: 1605-7422/13/$18 · doi: 10.1117/12.2004937
Proc. of SPIE Vol. 8581  85810K-1
Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 9/5/2018
Terms of Use: https://www.spiedigitallibrary.org/terms-of-use
Conical lens
Anesthesia gas tube
c - - Optical condenser
! Water
Transducer array
Animal
Heater
Flexible
membrane
agnetic base
512 preamps
8:1 multiplexers
64- channel
amplifier& DAQ
Ti- sapphire laser
1
Computer
,,,..,
1
 
 
signals. The photoacoustic signals were detected by a 512-element full-ring transducer array with 5 MHz central 
frequency (80% bandwidth) and 50 mm ring diameter. Each element in the array was mechanically shaped into an arc to 
produce an axial focal depth of 19 mm within the imaging plane. The combined foci of all elements generated a central 
imaging region of 20 mm diameter and 1 mm thickness [11, 12]. Since the light incidence was oblique, the light formed 
a weak focus inside the animal body. This focal region overlapped with the acoustic focal plane to improve the 
efficiency of detecting photoacoustic signals generated in deep tissues [7]. 
 
 
 
 
Figure 1. Experimental setup of the full-ring confocal whole-body photoacoustic computed tomography system. 
 
3. SYSTEM CALIBRATION 
In spectral experiments, the beam profile of the laser varied at different wavelengths, which led to changes in the fluence 
distribution, adding complexities on spectral separation. This problem can be solved either by aligning the optics after 
each wavelength tuning, which requires a tremendous amount of time in three-dimensional scanning, or more efficiently, 
by post-experimental compensation. In this research, we used a phantom calibration method to compensate for both the 
laser power and the light band uniformity of different wavelengths. The phantom was made from a gelatin-water 
suspension mixed with 0.1% black ink. The suspension was put in a cylindrical container, whose inner diameter was 
similar to the mouse cross-sectional diameter (20 mm), and was then cooled in the fridge until gelled. The gelled 
phantom was then mounted on the RC-PACT scanner and imaged using the same scheme as the animal experiment. 
Cross-sectional photoacoustic images of the phantom were reconstructed at each wavelength, and the ring-shaped light 
variation was obtained based on the surface signal of the phantom image. The matrix of light variation was then used as 
a weighting function to calibrate the spectral in vivo images. The calibration results are demonstrated in Figure 2. The 
compensated images have similar signal intensity at different wavelengths and a more uniform signal distribution around 
the illumination ring.  
Proc. of SPIE Vol. 8581  85810K-2
Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 9/5/2018
Terms of Use: https://www.spiedigitallibrary.org/terms-of-use
 
 
5 mm
(a) (b) (c)
(d) (e) (f)
(g) (h) (i)
776 nm 796 nm 820 nm
PA
 a
m
pl
itu
de
 (
a.
u.
)
Max
Min  
Figure 2. Image compensation for the wavelength-dependent laser power and ring-shaped-light distribution. Top row (a-c), un-
compensated images. Middle row (d-f), phantom images. Bottom row (g-i), compensated images. Left column: images acquired at 
776 nm. Middle column: images acquired at 796 nm. Right column: images acquired at 820 nm. 
 
4. IN VIVO ANATOMICAL IMAGING 
To demonstrate the in vivo anatomical imaging capability of our RC-PACT system, we imaged healthy athymic (nude) 
mice. Figure 3 shows serial cross-sectional images of 5-6-week-old healthy athymic mice in axial views. The brain 
cortex, heart, liver, spleen, and kidneys are clearly visible. Additionally, detailed vascular structures within these organs 
are visible, demonstrating that the system can be used for whole-body angiographic imaging without injecting 
exogenous contrasts. The spinal cord, stomach, and gastrointestinal tracts are imaged due to the surrounding 
microvasculature. Major blood vessels, such as the vena cava, are also clearly visible. Using exogenous optical contrast 
(e.g., near-infrared dyes), the system can also image organs with little blood. The bladder image was acquired 30 
minutes after tail vein administration of IRDye800-2DG. The urinary bladder showed strong contrast as it was filled 
with the dye excreted by the kidneys. 
Proc. of SPIE Vol. 8581  85810K-3
Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 9/5/2018
Terms of Use: https://www.spiedigitallibrary.org/terms-of-use
l 1
 
 
Liver
Heart
Brain
Kidneys
Intestine
Bladder
 
Figure 3. RC-PACT images of athymic mice acquired noninvasively at various anatomical locations: 
  
5. IN VIVO TUMOR GLUCOSE METABOLISM IMAGING 
To showcase the capability of our RC-PACT system to image the glucose metabolism of deeper tumors, we imaged two 
mice with orthotopically implanted 786-O tumor cells in their left kidneys.  
Following the control experiment, 200 µL of (0.4 mM) IRDye800-2DG was administered, and the mice were imaged 
after 24 hours. This 24-hour time window is sufficient for excess unbound IRDye800-2DG to be excreted by the kidneys 
and bladder. This also increases the likelihood that the imaged IRDye800-2DG reflects tumor glucose metabolism rather 
than the physical entrapment of excess dye molecules inside the tumors. Figures 4(a) show representative anatomical 
cross-sections of the two 786-O tumor mice. The HbT concentrations were normalized by the maximum HbT 
concentration (observed in the vena cava), scaled to 2.3 mM [Figures 4(b)], and then the absolute IRDye800-2DG 
concentrations in the anatomical cross-section were estimated. Images of tumor glucose metabolism [figures 4(c)] were 
overlaid on the gray-scale PACT images acquired at 776 nm. In Figures 4(c), the mean IRDye800-2DG concentration in 
healthy kidneys were used as the threshold to account for non-specific IRDye800-2DG uptake due to kidneys being the 
excretory route. The signal-to-noise ratio for imaging IRDye800-2DG in the most metabolically active site in the tumor 
was 39.3 dB for mouse 1 and 38.3 dB for mouse 2. The tumor-to-normal tissue contrast for IRDye800-2DG uptake was 
approximately 3.3 at the most metabolically active site in the tumor. The same image reconstruction and spectral 
separation methods were also applied on the control RC-PACT images, and no significant IRDye800-2DG uptake was 
observed. 
 
Proc. of SPIE Vol. 8581  85810K-4
Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 9/5/2018
Terms of Use: https://www.spiedigitallibrary.org/terms-of-use
Awl
PA am 1"rtu& (á.u.
(c)
[Hal ("RAI)
10 16 20 26 30
-2-r- -1 a 9 fir- 3
PA amplitude (a.u.)
AMIN=
12 lE3 "IS
PG11 (11M)
iF 04 1.2 14 2.1. 3.0 d.
[i113u J
 
 
 
 
 
Figure 4. In vivo RC-PACT images of orthotopically implanted 786-O kidney tumors. Top row: mouse 1. Bottom row: mouse 2. (a) 
Anatomical images acquired at 776 nm. (b) HbT images overlaid on the anatomical images. (c) IRDye800-2DG images overlaid on 
the anatomical images. CK, cancerous kidney; HK, healthy kidney; HM, hypermetabolic. 
 
  
6. DISCUSSION AND CONCLUSION 
Anatomical and metabolic imaging is of great importance due to its wide application in tumor biology, drug discovery, 
and early-stage cancer screening in both clinical and pre-clinical settings. In this report, we have demonstrated that RC-
PACT provided tumor glucose metabolism and anatomy images with ultrasound-defined spatial resolution in a single 
imaging modality. While PET-CT and PET-MRI have been well established in the field, RC-PACT is a promising and 
less expensive alternative with the advantages of non-ionizing laser radiation, non-radioactive optical contrast, and 
endogenous hemoglobin contrast. Additionally, image co-registration of anatomical and metabolic assessments is 
automatic. Besides imaging tumor glucose metabolism, RC-PACT can also benefit from near-infrared fluorescent dyes 
[8] and proteins [13] for deep molecular imaging along with detailed, high resolution anatomical structures. The 
possibility of multiplexing functional parameters in combination with anatomical imaging uniquely positions RC-PACT 
to have a broad impact in preclinical medical research and life sciences. Due to the fixed ring diameter of the current 
system, we only demonstrated 1 cm penetration depth; however, photoacoustic imaging at depth of 8.4 cm has been 
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reported [14]. Taking advantage of the full-ring light illumination, which doubles the imaging depth (~16.8 cm), RC-
PACT can potentially be scaled to image larger animals or even humans. 
7. ACKNOWLEDGEMENTS 
The authors appreciate Ms. Seema Dahlheimer’s close reading of the manuscript. This work was sponsored in part by 
National Institutes of Health grants R01 EB000712, R01 EB008085, R01 CA134539, U54 CA136398, R01 EB010049, 
R01 CA157277, R01 CA159959, and 5P60 DK02057933. L.W. has financial interests in Microphotoacoustics, Inc. and 
Endra, Inc., which, however, did not support this work. 
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Anatomical and metabolic small-animal whole-body imaging using ring-shaped confocal photoacoustic computed tomography

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