858142.pdf

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Combined optical- and acoustic-

resolution photoacoustic microscopy

based on an optical fiber bundle

Wenxin Xing, Lidai Wang, Konstantin Maslov, Lihong V.

Wang

Wenxin Xing, Lidai Wang, Konstantin Maslov, Lihong V. Wang, "Combined

optical- and acoustic-resolution photoacoustic microscopy based on an optical

fiber bundle," Proc. SPIE 8581, Photons Plus Ultrasound: Imaging and

Sensing 2013, 858142 (4 March 2013); doi: 10.1117/12.2005259

Event: SPIE BiOS, 2013, San Francisco, California, United States

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Combined

optical

and acoustic

resolution photoacoustic microscopy

based on an optical fiber bundle

Wenxin Xing

Lidai Wang

Konstantin Maslov

Lihong V. Wang

a,b

Optical

Imaging Laboratory, Department of Biomedical Engineering,

Preston M. Green Department of Ele

ctrical and Systems Engineering

Washington University in St. Louis, One Brookings Drive, St. Louis, Missouri 63130, USA

Corresponding author: lhwang@wustl.edu

ABSTRACT

Photoacoustic microscopy (PAM), whose spatial resolution and penetration depth are both scalable, has made great

progress in recent years. According to their different lateral resolutions, PAM systems can be categorized into either

optical

resol

ution (OR) PAM, with optical

diffraction

limited lateral resolution, or acoustic

resolution (AR) PAM, with

acoustically limited resolution and a deeper maximum imaging depth. In this report, we present a combined OR and AR

PAM system with resolutions of

2.2 μ m and 40 μ m, respectively

. Sharing most components between the OR and AR

implementations, the system achieves separated illumination for OR and AR imaging by an optical fiber bundle through

different channels, and two discrete lasers are used to prov

ide either high

power energy for AR imaging or high

repetition

rate pulses for OR imaging.

he design enables

automatically co

registered

OR and AR photoacoustic

imaging in one single system, which extends the usability of current photoacoustic systems and

simplifies the imaging

procedure.

Keywords:

Photoacoustic imaging

fiber bundle

INTRODUCTION

Due to its optical absorption contrast and high spatial resolution that is scalable with the maximum imaging

depth, photoacoustic microscopy (PAM) has been suc

cessfully applied to

in vivo

imaging at scales from organelles

to organs [1, 2]. According to their different lateral resolutions, PAM systems can be categorized into either

acoustic

resolution (AR) PAM or optical

resolution (OR) PAM. AR

PAM [3, 4], whose

lateral resolution is

determined by the acoustic focus, can achieve tens of microns lateral resolution with a maximum imaging depth of

several millimeters. OR

PAM [5, 6] has a tighter optical focus than acoustic focus and can achieve optical

diffraction

mited lateral resolution with a maximum imaging depth of ~1.2 mm [7]. With these complementary

characteristics, AR

and OR

PAM systems have been used in tandem to image samples in many applications [8, 9].

However, since the systems share similar optical a

nd ultrasonic components and designs, it is advantageous to

combine them, which would facilitate either sequential or simultaneous operation, yielding automatically co

registered images and also reducing system cost. Here, we present a method to

combine

and AR

PAM

systems based on an optical fiber bundle.

SYSTEM

DESIGN

A schematic of the system setup is shown in Fig. 1. A high

repetition

rate laser (SPOT 10

200

532, Elforlight)

provides photoacoustic excitation for OR imaging, whereas a high

power lase

r (Cobra, Sirah) pumped by a

Nd:YLF laser provides photoacoustic excitation for AR imaging. The high

repetition

rate laser o

perates at 532 nm,

near the 530

nm isosbestic absorption wavelength of oxy

and deoxy

hemoglobin, and it delivers light energy on

e tissue surface up to 0.1 μJ/pulse at 20 kHz. Because the high

power laser cannot operate at 532 nm, it is tuned

to another isosbestic point at 570 nm, where hemoglobin has comparable optical absorption with hemoglobin at

532 nm, and the delivered light e

nergy on the tissue surface is up to 60 μJ/pulse at 2 kHz.

merging from the

lasers

, both beams

are vertical

ly polarized.

e OR beam is focused by a condenser lens (LA1214

A, Thorlabs),

then spatially filtered by

diameter

inhole.

he AR beam

passes through a

beam expander

(OL1 and

OL2 )

with 1.5X magnification

and then

becomes horizontally polarized by reflection off

mirror

. The two

perpendicular

laser

beams are combined

a polarizing beamsplitter cube (PBS201, Thorlabs). Because of the

Photons Plus Ultrasound: Imaging and Sensing 2013, edited by Alexander A. Oraevsky, Lihong V. Wang,

Proc. of SPIE Vol. 8581 858142-1

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(a)

Computer

High -power

AR laser

High- repetition-

rate OR laser

OL1 OL2

DAQ

card

(c)

Motion

controller

Photoacoustic

imaging stage

To DAQ

'Amplifier

014,5

Prism

Sample

(a)

Computer

High -power

AR laser

OL1 OL2

DAQ

card

High- repetition-

rate OR laser

To DAQ

uAmplifier

Motion

controller

Photoacoustic

imaging stage

Motor stage

Sample

cube

’

s high extinction ratio (T

>1000:1

for transmission;

, R

~100:1

for

reflection; Subscript s: s

polarized

beam, i.e., perpendicularly polarized AR beam; Subscript p: p

polarized beam, i.e., parallel polarized OR beam

most of the beam energy goes

in the direction of a

fiber coupler

C1, Newport

with a 10X

magnificati

objective lens). After attenuation by a variable neutral density filter (NDC

100C

2, Thorlabs

, not shown

), the two

beams pass into

the

fiber coupler

A fiber bundle is used to achieve

switchable

AR and OR illumination (

IGN

037/10

Sumitomo

, core number

,000

, single c

ore

meter

: 2.0

, entire core area diameter ~ 350

). A

single core is used to deliver OR light in order to achieve a small spot size and hence high

lateral

resolution, while

the

entire core area is used to deliver more energy

for

illumination

. To focus the OR beam onto a single core of

the fiber bundle, the pinhole is located 20 cm from the fiber coupler. T

e fiber coupler also focuses

the

collimated

AR beam over the entire core area. The tip of the fiber bundle is aligned at the f

ocus of the OR beam, which is

slightly away

from the focus of the AR beam

[

shown in F

g. 1

(b)]

making it

match the entire core area

at the fiber

tip.

The other end of the fiber bundle is mounted to a photoacoustic

imaging

stage

[

Fig 1(c)

]

. The laser

beams

emerging from the fiber bundle pass through two identical optical condenser lenses, reflect off the aluminum

coated hypotenuse of a prism, and then focused into the sample. The generated photoacoustic signal is collected by

Figure

(a) Schematic of the combined AR and OR photoacoustic microscopy system. (b)The interior of the fiber coupler.

(c).Detailed design of the photoacoustic imaging stage.

All images are side view images. AL, acoustic lens;

DAQ,data

acquisition; FB, fiber bundle; FC, fiber coupler; M,

mirror; OL

optical lens; PBS

polarizing beamsplitter; PH

pinhole;

ultrasound transducer; WT

water tank.

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(a)

AMES

(b)

0.8

0.6

z0.4

0.2

200

0.5

-0.5

40μm

400

600

800

X [μm]

the acoustic lens, which i

s in confocal arrangement with focused laser beam, and then received by a 50 MHz

ultrasound transducer (V214, Olympus NDT). The electrical signal from the ultrasonic transducer is amplified,

digitized and analyzed by a personal computer, which also synchro

nizes the motion controller with the DAQ card

and the lasers. A more detailed description on fast voice

coil scanning can be found in our previous publication

[

]

Since the photoacoustic probe weighs less than 40 g as our previous one, the fast scanning ability is maintained in

the

combined

OR/AR

PAM system.

RESULT

3.1

System

resolution

To evaluate the performance of the system, the spatial reso

lutions for both OR and AR imaging were measured

A resolution target (1951 USAF Hi

Resolution Targets, Edmund Optics) was imaged in water with only OR

PAM

The system

can

resolve element 6 in group 8

[

arrow in Fig. 2(a

)

]

with

456

line pairs

per millimeter,

which

translates into

an OR imaging

lateral resolution

finer than

2.2

μm. The discrepancy from

the

theoretical resolution

of 1.4

μm

(NA: 0.2, wavelength: 532

) may be attributed to optical aberration and higher

order modes of the

optical

fiber.

The lateral resolution of

the

imaging

was evaluated by imaging a 6 μm

diameter carbon fiber as

an approximate

line

target

The

lateral

full width at half

maximum (FWHM) is 40 μm, as shown in Fig. 2

(

)

Since

the axial resolution is determined by

the acoustic parameters only, the OR and AR imaging share the same axial

resolution.

The aixal resolution was estimated by t

he shift

and

sum method

[

]

. It

simu

lates the situation when

two line sources are positioned close to each other.

The a

xial

line spread function

(

LSF

)

, its shift (

16 μm apart

) and

the

enve

lope of their sum

are

shown in

Fig. 2(c)

The LSF is acquired by averaging 300 measurements, so it can

be treated as a noise

free result.

The enve

lope of the sum

can be regarded as the envelope of the PA signal when

two line sources are 16 μm apart in the axial direction.

The

fact that the envelope has two peaks means two lines

can be resolved. However, the

actual resolving ability is always limited by noise. Here we define the contrast as

Figure 2 (a) OR

PAM image of a resolution test

target (b) Lateral LSF of the AR

PAM. (c) Axial LSF (

black

solid curve)

, its shift

(

red

solid curve)

and the

envelope

of their sum

(blue dashed

curve)

. NPA,

normalized

photoacoustic amplitude

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3

Depth [mmj

0 0

0

A W

X

3

Depth [mm]

0 O 0

A W N

..y s-,1

f~.- ,'`

,:" .

f;> .

`1/S

the signal difference between the bottom of the d

ip and the peak of the envelope,

and the noise as the standard

deviation of the signal noise in one single measurement.

e resolution ba

sed on the shift

and

sum method

is ~16

μm at a contrast

noise ratio of 2

3.2

In vivo

mouse imaging

The system enables

simultaneous

OR and AR imaging.

The

two lasers are triggered alternately, each firing at

its own pulse

repetition rate,

and

he OR laser fires with less energy than the AR laser. For highest resolution, the

OR laser is triggered with every step of the motor scanning, while the AR laser is triggered only once in ten steps.

Thus, t

he OR and AR images

can be

acquired simultan

eously in one single scan, with step sizes of 2 μm and 20

μm and laser repetition rates of 20 kHz and 2 kHz, respectively.

In vivo

mouse imaging was performed by the

combined system.

All experimental animal procedures were carried out in conformity with th

e laboratory protocol

approved by

the Animal Studies Committee at

Washington University in Saint Louis.

The mouse

(Athymic Nude

Mice, Harlan)

was anaesthetized by isoflurane and positioned on an animal platform.

The ear of a nude mouse

was imaged

both

PAM and OR

PAM

as shown

Fig. 3

The OR image shows fewer blood vessels due to

the penetration depth but better image due to

the

resolution. The arrow

in Fig 3(a) and Fig 3(b)

indicate

a deep

vessel that AR

PAM can image, but OR

PAM was unable to image. Fig 3(c) and Fig 3(d) show the cross

sectional

scan)

image located at dash

line position in Fig 3(a) and Fig 3(b). They clearly show that AR

PAM can image

deeper

than OR

PAM

ointed

by the small arrwos

in Fig 3(c) and Fig 3(d)

, AR

PAM can image the blood

vessels in both upper and

bottom

layer

of the mouse ear, hower OR

PAM can only image the upper layer.

The

large arrows mark the same vessel which is marked in Fig 3(a) and Fig

3(b). It confirms that OR

PAM is unable to

image it due to

its

shallow

penetration depth.

Figure 3 (a) O

PAM image of

the mouse ear.

(b)

PAM image of

the mouse ear. The

arrow

in (a) and (b)

indicate a vessel which was

successfully

imaged by

PAM only.

(c)

Cross

sectional (B

scan) OR image at

the position marked by dashed line in (a). The small arrow shows the upper layer of vessels. (d

)

Cross

sectional

scan) AR image at the position marked by dashed line in (b). The small arrows show bo

th the upper and

bottom layers of vessels. The big arrows in (c) and (d) indicate the same vessel as in (a) and (b).

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(d)

0.3mJ

--___---

(e)

0.3mJ

region on the back of a nude mouse was imaged

in vivo

as well. Fig 4(a) and Fig 4(b) show the OR and AR

image respectively.

Fig 4 (d) a

nd Fig 4 (e) show the cross

sectional image marked by the das

h line in Fig 4(a) and

Fig 4(b). Time

gain compensation was applied to show the deep signals better. AR

PAM is able to get signals up to

~ 1.5 mm

as the big arrow in Fig 4(e)

Since AR

PAM does n

ot have advantages over OR

PAM

shallow

depth

e remove the surface signal

0.5 mm depth)

the AR

PAM image

[

dash line area in

Fig 4(e)], and the new

image

is shown in Fig 4(c).

It clearly shows large deep vessel which is different from the vessels

shown in Fig 4(a).

merged

image with shallow OR image and deep AR image is shown in Fig 4(f).

It demestrate

complementary

information which the combined system can provide.

CONCLUSION AND DI

SCUSSION

To our knowledge, this is the first system that can perform OR and AR photoacoustic imaging simultaneously.

The method we present here requires only modest modifications to current widely used PAM systems. Not limited

to the two illumination choic

es introduced in this paper, the fiber bundle allows a wide range of illumination spot

sizes, from one single core to the entire core area, and optical scanning among the cores is also possible [12]. A

double clad [13] or multiple clad fiber may be applied

to provide flexible illumination spot sizes as well.

In this

report, we demonstrate that t

combined

system

can get

complementary

information when we

image

the sample

by OR

and AR

PAM

focused

at the same depth

, and remove the surface signal

acquired

PAM.

This

mode

Figure

In vi

vo image of mouse skin: (a) M

aximum amplitude projection

(MAP)

image by OR

PAM

(b)

MAP

image by AR

PAM

after the top 0.5mm surface signal removed (d) (e)

ross

sectional image at

the

dash

line marked position

in (a) and (b)

The

large arrow in (e) indicates that AR

PAM

could receive si

gnal up to 1.5mm depth in one single scan. Small arrows in (c) and (e) indicate the correspo

nding

blood vessels. Dashed line shows the shallow signal, which is removed in (c). (f) Overlaid

image of OR

image

and

surface

removed AR

image

Proc. of SPIE Vol. 8581 858142-5

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especially

beneficial in

the

imaging situations, when scans at the same depth are necessary. For example, scans by

and AR

PAM at the same depth are

very

useful for tumor imaging, since they can provide both strong

penetrating capab

ility for imaging a melanin

containing tumor and high

resolution capability for imaging the

blood vessels around the tumor.

Another mode is also

possible, by separating the optical focus and the acoustic

focus. Th

mode is suitable for

getting OR

and AR

PAM image

at different

depth

, and surface signal remove is

not

needed

[14]

In summary, the

combined

system has successfully achieved switchable spatial resolutions and maximum

imaging depths using an optical fiber bundle. This new

combined

OR/AR

PAM sy

stem can bring multiscale

imaging capability to many applications, such as anatomic imaging and label

free measurement of oxygen

saturation.

ACKNOWLEDGEMENTS

This work was sponsored by National Institutes of Health (NIH) grants R43 HL106855, R01 EB000712,

R01

EB008085, R01 CA134539, U54 CA136398, R01 CA157277, R01 CA159959, as well as the Cancer Frontier Fund.

L. V. Wang has a financial interest in Microphotoacoustics, Inc., and Endra, Inc., which did not support this work.

K. Maslov has a financial intere

st in Microphotoacoustics, Inc. which did not support this work. The authors

appreciate Prof. James Ballard’s close reading of the manuscript.

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