In-vivo real time non-invassive imaging for longitudinal translational research
Milan Kopecek
Regional Sales Manager
• Micro-ultrasound
• What is Micro-ultrasound?
• Visualization, measurement and analysis with the Vevo 2100
• Photoacoustics
• What is Photoacoustics?
• Visualization, measurement and analysis with the Vevo LAZR
• Applications
Agenda
Total Small Animal Solution
• Real-time
• Longitudinal Studies
• Measure Physiological parameters
• Contrast/Molecular Imaging
• Translatable to man
How Does Micro-Ultrasound Work?
How Does Micro-Ultrasound Work?
Micro-ultrasound (mouse fetus)
High-frequency = High-resolution
3 - 15 MHz
20cm
30 – 80 MHz
3cm
Melon
Coffee Bean
Conventional clinical ultrasound (human fetus)
Micro-Ultrasound: Clarity
200 – 300 micron resolution
30 micron resolution
Anatomy and Morphology
Cardiac Imaging: EKV
• High Temporal Resolution Left Ventricle in Adult Mouse at
10,000 frames per second
Visualization Measurement Analysis
• Tissue/organ/tumor identification
• Vasculature density, structure and flow
• Biomarkers
• Linear, area and volume measurements
• Blood flow velocity and vessel density
• Cardiac measurements
• Contrast Quantification including biomarker expression
• Heart wall motion and strain
Imaging with the Vevo
Visualization with Micro-Ultrasound
1 mm *WAP-TAg Mouse Model
Image courtesy of Cotarla I. and Furth, P., Lombardi Cancer Centre, Georgetown University
Early detection and Quantification of Pre-Palpable Mammary Tumors
Image courtesy of T. Minko, Rutgers, The State University of New Jersey
Video Loop
3D Quantification of Human Ovarian Tumor Model
Power Doppler Mode Imaging
Day 1 Day 4
PV = 3.18% PV = 11.22%
Images courtesy of Hastie, Chambers, Lacefield and Fenster; Robarts Research Institute, London
Precise interventions without the need for invasive surgery
Ultrasound Image-Guided Injections
Measurement with Micro-Ultrasound
Quantification of size, vasculature and perfusion
Early detection/screening
Perfusion Volumetrics
Vascularity
Cardiovascular measurements
LV analysis
Vascular stiffness
Blood flow
Strain
Analysis with Micro-Ultrasound
User defined regions of interest
Curve fit algorithms
Vevo MicroMarker Quantification
Velo
city D
isplacem
ent
Strain
S
train R
ate
Kovacs, Washington University
VevoStrainTM
• Micro-ultrasound
• What is Micro-ultrasound?
• Visualization, measurement and analysis with the Vevo 2100
• Photoacoustics
• What is Photoacoustics?
• Visualization, measurement and analysis with the Vevo LAZR
• Applications
Agenda
The Photoacoustic Effect
1) Nanosecond laser pulse illumination
3) Emitted pressure (sound) wave
4) Detection of ultrasound and creation of image
2) Optical absorption, heating and thermoelastic expansion
Optical Ultrasound
The Photoacoustic Effect
Light in
Sound out
5 mm 5 mm
5 mm
Meaningful images with the sensitivity of optical imaging and the resolution of ultrasound
The Photoacoustic Imaging
Visualization Measurement Analysis
• Blood signal
• Vascular density, structure
• Angiogenesis
• Oxygen saturation and total hemoglobin
• Photoacoustic signal intensity (contrast agent measurement)
• Absorption spectra
• Spectral unmixing
Imaging with the Vevo LAZR Photoacoustic System
Visualization with Photoacoustics
• Inherent co-registration of photoacoustic and anatomical images – Simultaneous registration of photoacoustic image on 2D and 3D planes – Real-time acquisitions for true in vivo monitoring
What is this signal?
Where is this signal?
Needle Skin Tumor Needle Skin Tumor
Delivery of nanoparticles into tumor
The Photoacoustic Imaging
Tumor vasculature imaging
Single wavelength imaging of endogenous signal
•Sensitive detection of blood signal
•Functional and non-functional vessels, blood pools
•2D and 3D
Measurement with Photoacoustics
( HbT = HbO2 + Hb )
Total Hemoglobin = Oxygenated Hemoglobin + Deoxygenated Hemoglobin
Near-Infrared (NIR)
Oxygen Saturation Calculation
0
10
20
30
40
50
60
70
80
90
0 100 200
Oxyg
en
Satu
rati
on
(%
)
Time (s)
Region 1 Region 2
100% O2 100% O2 5% O2
Change in Oxygen Saturation in Tumor
Region 1
Region 2
Analysis with Photoacoustics
Endogenous contrast
Contrast
Agent
Applications
Vevo LAZR Contrast Applications
Absorption Spectra Analysis – Spectro Imaging
Indocyanine green absorption spectra measured by photoacoustic imaging from 680 to 970 nm wavelengths.
Applications of Ultrasound and Photoacoustics
Single Walled Carbon Nanotubes conjugated to RGD peptide target αvβ3 integrins
(Gambhir lab, Stanford University)
(Heinmiller, et al. EMIM 2011)
Targeted carbon nanotubes
Change in Measured Photoacoustic Signal over time after carbon nanotube injection
Normal
Color Doppler Oxygen
saturation Total
hemoglobin
Stroke 2
Stroke 1
Stroke Imaging – Skin removed
Melanin
Oxygenated blood
3D Imaging of Human Finger
Pharmacokinetics with Photoacoustics
Identifying IR800-2DG in the kidney
Peak ~785nm Peak 760nm
Control and kidney
regions have peak PA
signal at different
wavelenths
Peak ~785nm
IR800-2DG in phantom
Spectral unmixing
Pre
24hrs post
6hrs post
Blue = DeoxyHb Red = OxyHb Green = IR800 2_DG
Quantifying IR800-2DG in the kidney
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Pre 6 hrs post 24 hrs post
3D quantification of PA signal in the kidney pre, 6hrs and 24hrs post-IR800-
2DG
780nm 850nm
Methylene blue i.v. Injection – kidney imaging • Subtraction image of bolus injection of methylene blue using the ‘invisible
table’ and trifurcated fiber
Methylene blue i.v. Injection – kidney imaging
Pre-bolus
7 mins Post-bolus
Methylene blue i.v. Injection – kidney imaging
Reporter gene imaging with Photoacoustics
Photoacoustic Imaging of an Inducible Reporter
• Tyrosinase, the enzyme responsible for melanin production was used as an inducible reporter
• Animals with TYR- and TYR+ tumors on each flank were imaged before and after induction of tyrosinase expression
Abs.
Figure modified from Murkin and Arango. Near-infrared spectroscopy
as an index of brain and tissue Oxygenation. Br J Anaesth 2009; 103 (Suppl. 1): i3–i13
-TYR
+TYR
Absorption spectra for hemoglobin and
melanin
Post-induction Pre-induction
TYR-
a)
Photoacoustic Imaging of an Inducible Reporter
TYR+
a)
Photoacoustic Imaging of an Inducible Reporter
a)
Multi-modality Molecular Imaging with Tyrosinase as a Reporter Gene
MRI PET
PA
Figures from Cheng, Z. et al. Tyrosinase as a multifunctional reporter gene for Photoacoustic/MRI/PET triple modality molecular imaging. Sci Rep. 2013 Mar 19;3:1490
Imaging Enzymatic Activity
Pre
Post
680 nm 750 nm
Min
Max Pre Injection
680-750
Post Injection
680-750
Subtraction of Images
Levi, J. et al. Clin Cancer Res, 2013
MSCs + NTs Control MSCs + NTs Control
MSCs + NTs
in vivo monitoring MSC’s labeled with nanotracers
Nam et al SPIE 2012
Ischemic muscle
PEGylated fibrin gel
MSCs labeled with
NTs
US/PA imaging
PEGylated fibrin gel with MSCs +
NTs (105 cells/mL)
MSCs+NTs
Skin
HbO2
Hb
Longitudinal in vivo monitoring MSC’s labeled with nanotracers
Nam et al SPIE 2012
PEGylated fibrin gel with MSCs +
NTs
DAY 0 DAY 3 DAY 10 DAY 7
Injection
3D combined ultrasound and spectroscopic images of the gastrocnemius in which the
PEGylated fibrin gel containing MSCs/NTs was injected (Day 3,7, and 10)
23 mm x 12.5 mm x 25 mm
Neuroimaging with Photoacoustics
Brain imaging with photoacoustics
• Mouse brain coronal section
Non-Invasive 2D Brain Imaging with Oxyhemo Mode – Prototype LZ201
PCA
PCommA.
ICA
ICA
Noninvasive imaging of mouse brain
From: Dorr, et al. Neuroimage, 2007
Non-Invasive 3D Brain Imaging with Oxyhemo Mode
Superior sagittal sinus
Inferior cerebral
vein
Skin intact Skin intact
OxyZated Oxygen saturation map
HemoMeaZure Total hemoglobin map
1mm 1mm
Imaging of mouse brain ventricles
LZ250 LZ550
Methylene Blue
Injection site
Imaging of mouse brain ventricles
MRI microscope Multiplexed Vevo LAZR
PA image showing methylene blue in the ventricles
Van Hagen et al., Neurobiology of Disease, 2007.
Brain Tumor Imaging with Microultrasound and
Photoacoustics
Swanson Biotechnology Center Animal Imaging and Preclinical Testing Facility
Scott Floyd lab
Nonlienar contrast imaging of perfusion with microbubbles
Nonlinear contrast Mouse #1
Images courtesy of: Swanson Biotechnology Center Animal Imaging and Preclinical Testing Facility and laboratory of Scott Floyd, Koch Institute for Integrative Cancer Research, MIT
Nonlienar contrast imaging of perfusion with microbubbles
Nonlinear contrast
Hypoperfused area
Mouse #1
Contrast quantifiation (relative blood volume)
Images courtesy of: Swanson Biotechnology Center Animal Imaging and Preclinical Testing Facility and laboratory of Scott Floyd, Koch Institute for Integrative Cancer Research, MIT
Multi-modality imaging of hemodynamics in the brain
Potential hypoxic area Mouse #1
Perfusion Flow Oxygenation Images courtesy of: Swanson Biotechnology Center Animal Imaging and Preclinical Testing Facility and laboratory of Scott Floyd, Koch Institute for Integrative Cancer Research, MIT
Multi-modality imaging of hemodynamics in the brain
Mouse #1
Perfusion (Relative blood volume, flow estimates)
Flow (Percent vascularity)
Oxygenation (sO2, HbT)
Images courtesy of: Swanson Biotechnology Center Animal Imaging and Preclinical Testing Facility and laboratory of Scott Floyd, Koch Institute for Integrative Cancer Research, MIT
Mouse #1
0
10
20
30
40
50
60
70
80
90
100
10 20 30 40 50 60
sO2
Avg
(%
)
Coronal slice # (caudal to rostral)
Avg sO2 in L and R Hemisphere (mouse 1) Caudal to rostral slices
L Hemisphere sO2
R Hemisphere sO2
Quantification of oxygen saturation in L vs R hemispheres
Images courtesy of: Swanson Biotechnology Center Animal Imaging and Preclinical Testing Facility and laboratory of Scott Floyd, Koch Institute for Integrative Cancer Research, MIT
Nonlinear contrast Mouse #2
Nonlienar contrast imaging of perfusion with microbubbles
Images courtesy of: Swanson Biotechnology Center Animal Imaging and Preclinical Testing Facility and laboratory of Scott Floyd, Koch Institute for Integrative Cancer Research, MIT
Nonlinear contrast
Hypoperfused area
Mouse #2
Hyperperfused area
Hypoperfused area
Contrast quantifiation (relative blood volume)
Nonlienar contrast imaging of perfusion with microbubbles
Images courtesy of: Swanson Biotechnology Center Animal Imaging and Preclinical Testing Facility and laboratory of Scott Floyd, Koch Institute for Integrative Cancer Research, MIT
Mouse #2 Potential hypoxic area
Perfusion Flow Oxygenation
Multi-modality imaging of hemodynamics in the brain
Images courtesy of: Swanson Biotechnology Center Animal Imaging and Preclinical Testing Facility and laboratory of Scott Floyd, Koch Institute for Integrative Cancer Research, MIT
Mouse #2
Perfusion (Relative blood volume, flow estimates)
Flow (Percent vascularity)
Oxygenation (sO2, HbT)
Multi-modality imaging of hemodynamics in the brain
Images courtesy of: Swanson Biotechnology Center Animal Imaging and Preclinical Testing Facility and laboratory of Scott Floyd, Koch Institute for Integrative Cancer Research, MIT
Mouse #2
0
10
20
30
40
50
60
70
80
90
100
10 20 30 40 50
sO2
Avg
(%
)
Coronal slice # (caudal to rostral)
Avg sO2 in L and R Hemisphere (mouse 2) Caudal to rostral slices
L Hemisphere sO2
R Hemisphere sO2
Quantification of oxygen saturation in L vs R hemispheres
Images courtesy of: Swanson Biotechnology Center Animal Imaging and Preclinical Testing Facility and laboratory of Scott Floyd, Koch Institute for Integrative Cancer Research, MIT
Stroke imaging with Microultrasound and
Photoacoustics
Stroke vs normal animal - quantification
0
10
20
30
40
50
60
70
80
90
Stroke 1 Stroke 2 Normal
sO
2 (
%)
3D oxygen saturation
0
5000
10000
15000
20000
25000
Stroke 1 Stroke 2 Normal
Hb
T (
a.u
.)
3D total hemoglobin
0%
2%
4%
6%
8%
10%
12%
14%
16%
Stroke 1 Stroke 2 Normal
PV
(%
)
3D percent vascularity (color Doppler)
Human imaging with Microultrasound and
Photoacoustics
Human Imaging
• Ultrasound/Photoacoustic transducer mounted to a 3D stepper motor
3D Imaging of Human Vasculature
• 3D MIP of the palm • 3D MIP of the forearm
• 3D MIP of the index finger
40mm
20mm
40mm
20mm
• 2D image of the forearm
Finger Imaging - Ischemia
• Dental floss tied around index finger to induce ischemia
• 2D imaging of finger ischemia – Oxyhemo mode
Finger Imaging - Ischemia
• 2D imaging of finger ischemia –Spectro mode
Abs.
Figure modified from Murkin and Arango. Near-infrared spectroscopy as an index of brain and tissue Oxygenation. Br J Anaesth 2009; 103 (Suppl. 1): i3–i13
Imaging of ???
Doppler imaging of dermatofibroma
Photoacoustic imaging of dermatofibroma
Photoacoustic Imaging Co-registered High-resolution High-sensitivity Real-time Oxygen saturation Molecular imaging In Vivo In Situ
LISTEN TO THE LIGHT
www.visualsonics.com
VisualSonics Insight through in vivo imaging
Head Office – Canada
3080 yonge street suite 6100
box 66 toronto canada M4N 3N1
US
100 park avenue, suite 1600
new york, NY 10017
Europe
Science Park 406
1098 XH AMSTERDAM
The netherlands
+1. 416.484.5000
+1.866.416.4636 (north america)
+800.0751.2020 (europe)
www.visualsonics.com
VisualSonics, VisualSonics logo, VisualSonics dot design, Vevo, Vevo MicroMarker, VevoStrain, VevoCQ, SoniGene, RMV, EKV, MicroScan, LAZRTight, Insight through In Vivo Imaging, are registered trademarks (in some jurisdictions) or unregistered trademarks of VisualSonics Inc. © 2011 VisualSonics Inc. All rights reserved.