Dr. Lawrence Yip explained how Photoacoustic (PA) imaging works, where it fits in with other modalities and, how your research could benefit from this emerging technology. Excellent spatial resolution, depth penetration, and superior contrast are just some of the advantages often associated with PA imaging. In this webinar, we dove into the advantages, where they can be beneficial, and how the TriTom’s patented technology overcomes some of the challenges experienced by early adopters of this imaging modality. The TriTom is a turnkey, compact, tabletop imaging system that combines the sensitivity of fluorescence molecular tomography with the depth penetration and spatial resolution of PA tomography. Many applications including cancer, neuroimaging, developmental biology, and cardiovascular research could benefit from adding these imaging modalities, and we will draw from literature and concrete examples to demonstrate this advantage.

1. An Introduction to
Photoacoustic Imaging
Featuring the TriTom™ in-vivo
imaging platform
September 20, 2023
Lawrence Yip PhD, Product Manager
lyip@scintica.com

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Topics of Discussion
• Motivation for photoacoustic imaging (PAI)
• Overview of the modality
• PA and fluorescence tomography as complementary modalities
• Preclinical applications
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Selecting a Modality
Sensitivity
PAI

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Motivation for PAI
• In vivo functional imaging analogous to functional MRI
• In vivo metabolic imaging analogous to PET
• In vivo label-free histologic imaging of cancer without excision
• In vivo molecular imaging of disease markers

5. Audience Poll

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Topics of Discussion
• Motivation for photoacoustic imaging
• Overview of the modality
• PA and fluorescence tomography as complementary modalities
• Preclinical applications
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Photoacoustic Tomography (PAT)
N-shaped pulse
Amplitude: absorption of light
Width: size of object
Includes elements from J. Lee et al., (2012) J. Breast Cancer

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Photoacoustic Tomography (PAT)
Biswas, D., Roy, S. & Vasudevan, S. Biomedical Application of Photoacoustics: A Plethora
of Opportunities. Micromachines 13, 1900 (2022).
A typical PA signal from circular numerical
target where a is amplitude, T is time of flight,
τ is width and χ is the relaxation time of the
PA time domain signal.

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Optical Absorption Spectra
Scholkmann, Kleiser, Metz, et al., (2014) NeuroImage; Zackrisson and Gambhir, (2014) Cancer Res

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Array Geometries
LCM Yip et al., "Approaching closed spherical, full-view detection for photoacoustic tomography," J. Biomed. Opt. 27(8)
086004 (2022) https://doi.org/10.1117/1.JBO.27.8.086004

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Topics of Discussion
• Motivation for photoacoustic imaging
• Overview of the modality
• PA and fluorescence tomography as complementary modalities
• Preclinical applications
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Fluorescence Imaging (FLI)
• Fluorescent molecules absorb light and are excited to a higher
electronic state
• The additional energy can be released as photons at a different
(longer) wavelength.
• Widely used, both for whole-animal imaging and in microscopy
Vilber Newton 7.0 FT500 Brochure

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FLI vs PAI
• FLI typically has higher molecular sensitivity than PAI due to the lack of
background signal from endogenous chromophores
• FLI has excellent spatial resolution for superficial tissue but degrades rapidly
with depth
Fluorophores generate both fluorescence emission and PA signals when
excited by the same nanosecond laser pulse
Together, FLI provides high sensitivity while PAT provides deep,
high-resolution imaging of fluorophores.

14. Audience Poll

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TriTom Technology
• Compact tabletop design
• Integrated gas anesthesia line
• Temperature-controlled imaging
chamber
• High-sensitivity sCMOS camera for
optical imaging
• Standard emission filters covering
popular fluorophores
• PA excitation from visible to NIR II
range
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TriTom™ small animal imaging platform

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TriTom Technology
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TriTom composite imaging of blood vasculature and
regional lymphatics in a live mouse model of
metastatic breast cancer.
Red – 895 nm excitation (deep vasculature)
Yellow – 532 nm excitation (superficial vasculature)
Scanned
region
True 3D with sub-millimeter spatial resolution:
160 µm x 160 µm in transverse planes
160 µm x 470 µm in sagittal and coronal planes
36 sec per multi-modality molecular imaging scan

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Quick & Easy Sample Handling
• Reliable Gas Anesthesia:
• Delivered through the hollow shaft
• Bite bar within nose cone secures position
• Convenient and Repeatable Procedure:
• Mouse’s legs and head are fixed in a
consistent position with minimal stress
• Preparation time < 5 min
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Mouse Holder –In Vivo Imaging
(1) Hollow shaft to fix the mouse holder inside
TriTom while reliably delivering anesthesia gas
(2) Bite bar for secure and repeatable mouse
positioning
(3) Plastic support rods
(4) Cushioned paw mounts
(5) Adjustable support block for mouse’s hind legs
1
2
3
4
5

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High Efficiency Optical Excitation
• Wavelength range
• All popular visible (460-659 nm)
• Standard NIR I/II excitation (650-1320 nm)
• Extended NIR II excitation (1065-2300 nm)
• High-energy (>350 mJ) 1064 nm mode
• High-resolution deep anatomical images
• 20 Hz pulse repetition frequency
• Fast wavelength switching (each pulse, arbitrary sequence)
• Excitation linewidth < 0.5 nm (equivalent to 1,280 excitation filters
• Integrated energy meter (quantitative imaging)
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Excitation Source: Tunable Pulsed Nd:YAG Laser

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Topics of Discussion
• Motivation for photoacoustic imaging (PAI)
• Overview of the modality
• PA and fluorescence tomography as complementary modalities
• Preclinical applications
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Applications
• Anatomical Imaging
• Cancer
• Contrast Agent Development
• Tissue Engineering &
Regeneration
• Neuroimaging
• Developmental Biology
• Therapeutic Monitoring
• Molecular Imaging
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21. Audience Poll

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Anatomical Imaging
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High energy 1064 nm excitation of mouse
thorax and upper abdomen. (Click video to
play)
(1) Heart; (2)Liver; (3) Aorta; (4) Spleen;
(5) Left Kidney; (6) Right Kidney; (7)Intestines.
Whole-body imaging Deep-tissue structures
800 nm PAT reconstruction of a nu/nu nude
mouse
Kidney; Scale bar – 1
mm
1 – abdominal aorta
2 – interior vena cava
3 – renal vein
4 – renal cortex
5 – renal artery
6 – interlobular artery
(vein)
7 – medulla (renal
pyramids)
Superior Anatomical Reference

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Cancer
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Monitor Tumor Growth, Metastasis, and Microenvironment
Human breast ductal carcinoma
xenograft (BT474 cells) in nu/nu
mice
(Left) Maximum intensity projection
of 532 nm (yellow) and 890 nm
(red/grey) showing superficial and
deep tumor vasculature
(Right) Composite skin (532 nm;
grey) and deep tissue (890 nm; red)
3D images.
Tumor size = 10.6 x 4.7 x 11.6 mm3
Tumor
Dumani et al, Proceedings SPIE 10878, Photons Plus Ultrasound: Imaging and Sensing, 108784Y (2019)

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Regional Lymphatic Drainage
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Tracking Metastasis Development
Normal mouse, 24-hrs post-injection
Subcutaneous injection of glycol-chitosan-coated gold nanospheres (50 µg/ml) mixed with ICG (50 µg/ml)
Total injection volume 50 µl
Fluorescence image
Injection site
Lymph node
Lower abdomen of a healthy
mouse
Red – deep blood vessels,
890 nm excitation scan
Yellow – superficial
vasculature (skin), 532 nm
excitation scan
1 – right subiliac lymph node
2 – injection site (right
mammary fat pad)
1
2
Photoacoustic image

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Quick & Easy Sample Handling
• High Throughput:
• Interrogate up to 10 samples per scan
• Preparation time < 5 min
• Small Sample Volume:
• 50 μL sample volumes
• Convenient and Repeatable Procedure:
• Radially oriented slots consistently hold samples in
place
• Allows for quick setup and removal of cuvettes
• Cuvette holder is instantly placed inside the TriTom
via magnetic connections
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Cuvette Holder – Development of Contrast Agents
(1) Port for administering liquid scattering background
(2) Plastic support rods
(3) Radial slots for quick setup and removal of cuvettes
1
2
3

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Contrast Agent Development
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Phantom T1 T2 T3 T4
3-IR800 Control IR800-A (200 µM) IR800-B (100 µM) IR800-C (50 µM)
4-IR800 Control IR800-D (25 µM) IR800-E (12.5 µM) IR800-F (6.25 µM)
760 nm 780 nm 800 nm
Phantom
3
Phantom
4
Cross-sectional (axial) views of 0.8 mm tubes with IRDye800CW samples
3D PAT reconstruction of
Phantom 3; 780 nm excitation.

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Contrast Agent Biodistribution
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Molecular unmixing of an ICG-based contrast agent (green) overlaid on the 800 nm excitation scan of anatomy (white)
at 0, and 90-min post-injection. Circulation half-life of ICG contrast agent compared to ICG.
t = 0 min t = 90 min
Circulation
Half-life
Singh, S., et al., (2023). Size-tunable ICG-based contrast agent platform for targeted near-infrared photoacoustic imaging.
Photoacoustics, 29. https://doi.org/10.1016/j.pacs.2022.100437

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Tissue Engineering & Regeneration
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STEM Cell Tracking and Therapy Monitoring
3D photoacoustic image of gold nanosphere-labeled mesenchymal stem
cells locally injected in a rat spinal cord. The linear relationship between PA
signal and injection volume demonstrates the ability to track and monitor
cell migration in vivo. Scale bar = 10 mm.
Axial View Coronal View
The TriTom’s submillimeter spatial resolution in all three
anatomical planes makes image analysis and quantification easier
for applications with hard to identify anatomy.
Donnelly, E. M., et al., (2018). Photoacoustic Image-Guided Delivery of Plasmonic-Nanoparticle-Labeled Mesenchymal Stem
Cells to the Spinal Cord. Nano Letters, 18(10), 6625–6632. https://doi.org/10.1021/acs.nanolett.8b03305

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Neuroimaging
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10 mm-thick (A) coronal and (B) transverse maximum intensity projection
slabs of a PAT volume reconstructed from the 750 nm scan. 1) Superior
sagittal sinus, 2) transverse sinus, 3) confluence of sinus, 4) cerebral artery,
5)auricular artery, 6) jugular vein, 7) brachial artery, and 8) ophthalmic
artery. The scale bar is 5 mm in length.
PAT composite image of the upper torso
and brain of a female BALB/c mouse
acquired post-mortem with 532 nm
(orange) and 750 nm excitation (gray). (Click
video to play)
A B

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Developmental Biology
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Monitor Placental Function and Detect Pathologies in Pregnancy and
Development
Reconstructed PAT images of a pregnant mouse on gestational day 12 in (A) superficial, (B) deep tissue, and (C) composite of
superficial (orange) and deep tissue (gray) images. D) Reconstructed PAT images of a pregnant mouse on gestational day 17 in
deep tissue. The 1) common iliac arteries, 2) placentas, and 3) abdominal aorta are labelled. The scale bar is 10 mm in length.
A B C D
Huda, K., Wu, C., Sider, J. G., & Bayer, C. L. (2020). Spherical-view photoacoustic tomography for monitoring in vivo placental
function. Photoacoustics, 20, 100209. https://doi.org/10.1016/j.pacs.2020.100209

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Developmental Biology
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Reconstructed PAT images of a pregnant mouse on gestational day 17 (A) pre-injection, (B) 5 minutes post-
injection, and (C) 90 minutes post-injection of FA-PEG-ICG. The scale bar is 10 mm in length and the 1)
placenta and 2) spiral artery are labelled.
A B C
Huda, K., Wu, C., Sider, J. G., & Bayer, C. L. (2020). Spherical-view photoacoustic tomography for monitoring in vivo placental
function. Photoacoustics, 20, 100209. https://doi.org/10.1016/j.pacs.2020.100209

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Therapeutic Monitoring
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Deep tissue (808 nm) PAT reconstruction of the abdomen of a pregnant mouse
(A) pre-injection and (B) 13 minutes post-injection of G protein-coupled
estrogen receptor (GPER) agonist, G1. 1) Denotes the iliac artery and 2) denotes
the placenta. The scale bar is 10 mm in length.
A B
Huda, K., Lawrence, D. J., Lindsey, S. H., & Bayer, C. (2022). Photoacoustic tomography to assess acute vasoactivity of
systemic vasculature. Proceedings of SPIE, 11960, 1196007. https://doi.org/10.1117/12.2612093

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Molecular Unmixing
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NiSO4
CuSO4
720 nm 810 nm
The samples were scanned at A) 720 nm and B) 810 nm before the
unmixing algorithm was applied. The samples containing C) NiSO4 and
D) CuSO4 are shown after the unmixing algorithm was applied.
A B
C D
NiSO4
[3:1] NiSO4:CuSO4
CuSO
4
[1:1] NiSO4:CuSO4
[1:3] NiSO4:CuSO4
Sample NiSO4 (%) CuSO4 (%) CNR
[0:1] NiSO4 : CuSO4 0 100 3.08
[1:3] NiSO4 : CuSO4 25 75 2.74
[1:1] NiSO4 : CuSO4 50 50 2.23
[3:1] NiSO4 : CuSO4 75 25 2.96
[0:1] NiSO4 : CuSO4 100 0 2.73

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Q&A Session
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INFO@SCINTICA.COM
Please enter your questions
in the Q&A section.
Thank You!

35. Globally linking scientists with
precision tools for research
through expertise in science,
engineering and support

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TriTom Patented Technology
Patents:
• USA –US 10,709,333
16/897,506
• China –CN 201780057506.8
• EU –EP 3,488,224
• Germany -602017039807.1
• Canada –CA 3,031,905
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TriTom vs Alternative Platforms
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TriTom Whole-body PAT Reconstructions
Subcutaneous imaging
532 nm
Deep-tissue imaging
800 nm

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Outstanding imaging specifications
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TRITOM compared to state-of-the-art whole-body photoacoustic and optical small animal imaging systems
Parameter TriTom Photoacoustic Optical FMT
Company(s) PhotoSound iThera Medical Perkin Elmer
Vilber
Perkin Elmer
TriFoil
Model(s) TriTom Premium MSOT inVision 512-echo IVIS SpectrumCT
Newton 7.0 FT500
FMT 4000
InSyTe FLECT/CT
High-throughput
(<5 min/animal total)
yes no yes no
FL / PA excitation range (nm) 460 - 1300 660 - 1300 415 – 850
420 - 800
635, 670, 745, 790
642, 705, 730, 780
Excitation line width (nm) < 0.5 < 0.5 30
30
< 3
< 3
Skin-level res (mm) (b)
Axial / Coronal / Sagittal
PA: 0.16 / 0.47 / 0.47
FL: 5 / 0.1 / 0.1
PA: 0.15 / 5 / 5
FL: n/a
PA: n/a
FL: 5 / 0.1 / 0.1
PA: n/a
FL: 2 / 0.3 / 0.3
Deep-tissue res, >2 mm depth (mm)
(b)
Axial / Coronal / Sagittal
PA: 0.16 / 0.47 / 0.47
FL: 5 / 5 / 5
PA: 0.15 / 5 / 5
FL: n/a
PA: n/a
FL: 5 / 5 / 5
PA: n/a
FL: 2 / 2 / 2
Registration over hard-tissue
anatomy (bone/cartilage)
n/a yes (IVIS)
no (Newton)
no (FMT 4000)
yes (InSyTe)
Registration over soft-tissue
anatomy
All views: skin, blood vessels, blood-
rich organs
Axial view only: skin, blood vessels,
blood-rich organs, B-mode US
Skin
Functional (physiological) imaging
without contrast agents
Available with spectral unmixing for blood content, perfusion and
hypoxia (sO2), water and lipid content
n/a
Molecular imaging sensitivity High Medium High Medium
Molecular imaging unmixing Accurate, quantitative, various types
of molecular sensors
Accurate, quantitative Qualitative Less accurate, quantitative
Bioluminescence Hardware enabled n/a yes n/a

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TriTom:
Photoacoustic & Fluorescence Volumetric Imaging
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TriTom™ small animal imaging platform
• The TriTom integrates photoacoustic and fluorescence
tomography to enable multimodal 3D imaging of small
animal models in vivo
• High sensitivity co-registered imaging due to
simultaneous PA and FL excitation using a high-efficiency
tunable laser
• Anatomical, functional, and molecular imaging with
submillimeter resolution in deep tissue in a single
benchtop instrument
• Molecular imaging platform optimized for longitudinal
studies

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Neuroimaging
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2D Transverse slices of the 750 nm
PAT scans acquired at several
vertical displacements. 1) Arms, 2)
cerebellum, 3) auricular artery, 4)
cerebral artery, 5) medulla, 6)
outline of brain, 7) transverse
sinus, 8) confluence of sinus, 9)
sublingual vein, 10) facial vein, 11)
superficial temporal vein, 12)
subarachnoid space, 13) right eye,
14) left eye, and 15) optic track.
The scale bar is 5 mm in length.
A B
D E
C
F

41. Audience Poll