Deep-Tissue Imaging by Listening to Molecular

Vibration

Ji-Xin Cheng Biomedical Engineering, Chemistry,

Center for Cancer Research

Purdue University

jcheng@purdue.edu

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