deep-tissue imaging by listening to molecular...
TRANSCRIPT
Deep-Tissue Imaging by Listening to Molecular
Vibration
Ji-Xin Cheng Biomedical Engineering, Chemistry,
Center for Cancer Research
Purdue University
Need for Molecular Diagnosis
Angiography Arterial Imaging Plaque Diagnosis
Though diseases are driven by altered molecular pathways, Current tools lack ability to identify chemical composition
Signals in CRS microscopy are generated by ballistic photons under the tight focusing condition, thus limiting the imaging depth to ~ 100 m.
Cover: multimodal image of central nervous system
TPEF
CARS Adventitia
Media
Lumen
Limited Penetration Depth in CARS Microscopy
Wang et al. ATVB, 2009
Time
= Distan
ce
0
Speed in soft tissues ~1500 m/s
Han-Wei Wang et al. Phys Rev Lett 2011, to be published.
RPL 106, 238106 (2011)
Principle of Photoacoustic Imaging
Laser
Photoacoustic Effect
Acoustic Detection
Pressure wave generation
Absorption, thermal-elastic expansion
Pulsed radiation transducer
Lihong Wang and coworkers, 2006 Nat. Biotech.
Blood Vessel Plexus (Hemoglobin) & Melanoma –
Po
int
scan
Stimulated-Raman Induced Photoacoustic Imaging
2007 to 2009 in Cheng lab, laser provided by Bob Lucht
fs pulse terahertz (no penetration through tissue) ns pulse ultrasound (1 to 100 MHz)
Contrast Ex. λ (nm) Absorption Cross-section
Sample Molar Density
VPA of CH (2nd Overtone)
1200 nm for CH stretching
~3x10-23 cm2/molecule
Olive oil ~5 x101 M, CH stretch bonds
PA of Hb (Electronic)
555 nm for Hemoglobin
9.1x10-17 cm2/molecule
Blood ~1.02 x10-4 M; 1.73 g/L in male blood
Comparison of energy requirements
106
105
Overtone Excitation Provides a “loud” Sound
Cias, Pawel, et al., Applied Spectroscopy, 61, 230, (2007) Telmissani, et al., Lab. Hematology, 5, 149, (1999)
CS2
Contrast Sample Ex. λ (nm) Adjusted Sig. Level
Ex. energy
Dichroic refl. factor
Obj. T%
Energy
VPA of CH (2nd Overtone)
Olive oil
1200 for CH stretching
3.4 V, P-P ~ 55 μJ ~57% ~70% 22 μJ
PA of Hb (Electronic)
Blood 555 for Hemoglobin
3.2 V, P-P ~ 10 μJ ~47% ~55% 2.59 μJ
• To produce the same level of signal, VPA imaging of olive oil using 2nd overtone of CH requires 10 times higher energy than PA imaging of blood using electronic absorption. • NIR excitation avoids photodamage.
Experiment results
Energy Requirement of Photoacoustic Imaging based on
Overtone Excitation
7000 8000 9000 10000
Am
plit
ud
e
Wavenumber (cm-1)
Wavelength (nm)1429 1250 1111 1000
a
b
10 100 1000 1000010
1
102
103
104
PA
Sig
nal (a
.u.)
Pulse Energy (a.u.)
c 11 12 13 14 15 16 17-2.4
-1.6
-0.8
0.0
0.8
1.6
2.4
3.2
Am
plit
ude
(V
)
Time (s)
Raw
Hilbert Transform
Butanal
PA signal waveform
Overtone Absorption
3n (2nd overtone absorption)
PA Signal from Vibrationally Excited Butanal Molecule May 2009
Fundamental: n=0 to 1 1st overtone: n=0 to 2
Molecular Vibration Mechanical Vibration
Fat
Collagen
Blood
Water
6000 7000 8000 9000 10000 110000.0
0.5
1.0
1.5
2.0
PA
Sig
nal A
mplitu
de (
V)
Wavenumber (cm-1)
Wavelength(nm)
1667 1429 1250 1111 1000 909
2nd overtone of
CH bond stretch
1st overtone and
combination of OH
stretch Collagen
PA Spectra of Biological Molecules
3rd overtone of
CH bond stretch
PRL 2011, 106, 238106.
x
y z
x
z y
Lumen
3.15 mm
Lipid core 250
200
150
100
50
0
PA Imaging of Lipid-rich Plaque
0 1 2 3 4 5 6 7 8 90.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
No
rma
lize
d V
PA
Sig
. In
t.
Thickness of Collagen Matrix (mm)
1/e 7 mm
Penetration Depth: up to 7 mm
Atherosclerotic Artery
VPA image
Speed: single pulse per pixel Spatial resolution: • Lateral resolution: from 5 m to 70 m • Axial resolution: ~135 m; 35 m is possible
PRL 2011, 106, 238106.
Input: 1064 nm
M1
M2
M3 M4
M5
M6
M7 M8 M9
M10
Output: 1197 nm or 1726 nm
Telescope
Isolator
HWP PBS
HWP
Ba(NO3)2
Beam trap
(B)
Output: 1064 nm
(C)
PH QR
Amp 1
λ/4
Amp 2
PH
FA AOM DL OI fiber
10
64
nm
11
97
nm
Stimulated Raman scattering
Ω = 1047 cm-1
(A)
2-kHz Raman Laser
Seeing the invisible
Side viewing fiber
Transducer
Intravascular photoacoustic (IVPA) 4 frames / s
IVUS IVPA Merged
Collaborators: Qifa Zhou (USC); Zhongping Cheng (UC Irvine); Michael Sturek (IUPUI)
Pu Wang et al. manuscript in preparation.
Imaging Plaque in the Presence of Blood
X
Z
1730 nm
1210 nm 255
0
Pu Wang et al, Journal of Biophotonics, 5: 25-32 (2012)
Hyper-spectral VPA Imaging of Fat and Collagen Pu Wang et al, J Biomed Opt 17(9), 096010 (2012)
Multivariate Curve Resolution – Alternating Least Squares (MCR-ALS) Analysis of Hyper-spectral VPA Image Pu Wang et al, J Biomed Opt (2012)
Vibronix Inc: Saving Lives by Label-free Imaging Technology
Figure 7: Catheter (A)
Imaging head
Catheter housing
Telescoping section
Luer connector
(C)
RO marker
Imaging window
PA/US probe Flexible shaft
(B) Fiber tip
Transducer
Catheter for Intravascular PA/US Imaging
Bond-selective Imaging of cm Deep Tissue: Vibration-based photoacoustic tomography
(VPAT)
Energy density (fluence) versus depth by Monte Carlo simulation
dermis layer μa=0.11cm-1 μs'=2.18cm-1
at 800 nm μa=0.13 cm-1 μs'=1.65 cm-1
at 1200 nm)
J Phys Chem Lett, 2013, 4: 3211-3215.
Proof of VPAT
J Phys Chem Lett, 2013, 4: 3211-3215.
0 10 20 30 40 50-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
-0.2
-0.1
0.0
0.1
0.2
0.3
Signal from target
PA
am
pli
tud
e (
a.u
.)
t (s)
Signal from chicken
VPAT imaging of fresh carotid artery from pig
• 128 elements, 10 frames per second, at 1210 nm.
1080 1120 1160 1200 1240 1280
35
40
45
50
55
60
65
PA
am
pli
tud
e (
a.u
.)
Wavelength (nm)
In collaboration with Craig Georgen (Purdue) and Michael Sturek (IUPUI)
VPAT Imaging of Intact Mouse Brain
Transducer of 128 elements, 10 frames per second, ex. at 1210 nm.
Star Trek, 1978 Paramount Pictures Corp.
McCoy: "Damn it Jim, I'm only a doctor!"
Function
Generator
Delay
Generator
Laser Beam
L
M
T
Vevo2100 System
3D
Nd: YAG
Pumped OPO
3mm
Schematic of VPA imaging probe and imaging set-up. L, f=250mm lens; M, reflection mirror; T, ultrasound transducer.
Hui Jie et al, manuscript in preparation.
P1
P2
P3
P4
PA 1210nm
PA 1110nm
a
PE Tube
US b
c 0
256
Chicken Breast
d
a
e
US b x
Z λ
Femoral Artery
Chicken Breast
f g h
Background
PA Image Stack
US/PA imaging of a highly diseased human femoral artery
above chicken breast and MCR-ALS resolved results
c d Blood Fat
Label-free Imaging Modalities
p
s
Raman/CARS/SRS
p as=2p-s
Photothermal
imaging
p
pr
n2
n1 ΔT
FTIR or transient
absorption
pr p
photoacoustic
Why Absorption ?
• More general than emission. Non-fluorescent material could be detected. • More sensitive to small objects absorption: ~ d3
scattering: ~ d6
Tcherniak & Link et al., Nano Lett, 2010, 10, 1398
t ΔT0 Probe
Pump
t
t t
Transient Absorption: Imaging Chromophores Having Undetectable Fluorescence
Two photon absorption Stimulated emission Excited state saturation Ground state depletion
Ultra-short pulses
Warren Warren, Duke University
Phase-sensitive Transient Absorption Imaging of Metallic and Semiconducting Single-Walled Carbon Nanotubes
b
(1) (2) (3) (1) (2) (3)
+1
0
-1
c
0.0
0.5
1.0
1.5
He
igh
t (n
m)
a
Phys Rev Lett, 2010, 105: 217401
SWNT
Fe padQuartz
Semiconducting Metallic
(d) (e)
C2
C1
V1
V2
S-SWNT
En
erg
y
Density of States
Ab
so
rpti
on
Wavelength
With out pump
With pump11
MEM-SWNT
10 μm
24 h
Speed: 2 s / pixel Green: Semiconducting SWNTs Red: Metallic SWNTs
In liver
5 μm
2012 Jan, 7: 56-61
In blood
tim
e
In-p
has
e
Qu
adra
ture
3 µm nanotubes Red blood
cells
Beating the Diffraction Limit: Saturated Transient Absorption Imaging of Non-fluorescent Species
Probe (ωpr)
Pump (ωp)
Saturation (ωsat)
t
At the very center of the focal spot
At the doughnut region (c)
t T0
T’ 1
t t
t t
Un-modulated
Modulated
Pump
Saturation
Probe
ωsat
ωpr
ωp
(b) (a) Probe only Pump-Probe (un-saturated)
Pump-Probe (saturated)
L1
L0
Saturated Transient Absorption
Saturated Transient Absorption Imaging of Nano-Graphite with Superior Resolution
Scale bar: 1 µm