Evans blue dye-enhanced capillary-resolution photoacoustic microscopy in vivo

Evans blue dye-enhanced capillary-resolution

photoacoustic microscopy

in vivo

Junjie Yao

Konstantin Maslov

Song Hu

Lihong V. Wang

Washington University in St. Louis

Optical Imaging Laboratory

Department of Biomedical Engineering

One Brookings Drive

St. Louis, Missouri 63130

Abstract.

Complete and continuous imaging of microvascular net-

works is crucial for a wide variety of biomedical applications. Pho-

toacoustic tomography can provide high resolution microvascular im-

aging using hemoglobin within red blood cells

RBCs

as an

endogenic contrast agent. However, intermittent RBC flow in capillar-

ies results in discontinuous and fragmentary capillary images. To over-

come this problem, we use Evans blue

dye as a contrast agent for

in vivo

photoacoustic imaging. EB has strong optical absorption and

distributes uniformly in the blood stream by chemically binding to

albumin. With the help of EB, complete and continuous microvascu-

lar networks—especially capillaries—are imaged. The diffusion dy-

namics of EB leaving the blood stream and the clearance dynamics of

the EB-albumin complex are also quantitatively investigated.

2009

Society of Photo-Optical Instrumentation Engineers.

DOI: 10.1117/1.3251044

Keywords: photoacoustic imaging; Evans blue dye; diffusion dynamics; Evans blue-

albumin clearance

Paper 09184R received May 9, 2009; revised manuscript received Aug. 28, 2009;

accepted for publication Sep. 2, 2009; published online Oct. 26, 2009.

1 Introduction

In the last decade, photoacoustic

tomography, which

combines the spatial resolution of ultrasound imaging with the

contrast of optical absorption in deep biological tissues,

has

gained great attention in biomedical applications.

Ultrasonic

imaging yields better spatial resolution in deep tissues than

optical techniques, because ultrasonic scattering is much

weaker than optical scattering,

making possible image recon-

struction based on propagated waves rather than energy diffu-

sion. However, pure ultrasonic imaging is insensitive to early

stage tumors and other biochemical properties, such as oxy-

gen saturation or concentration of hemoglobin, because it is

based on the detection of the bulk mechanical properties of

biological tissues. By combining optical imaging with ultra-

sound, PA imaging can achieve both high contrast and high

spatial resolution. PA imaging has been used for both struc-

tural and functional imaging of tumor,

brain cortex

perfusion,

microvascular structure,

and hemoglobin con-

centration and oxygenation,

as well as for lymph flow

cytometry.

Furthermore, it shows potential for blood flow

measurement.

Since the amplitude of the PA signal is pro-

portional to the absorbed optical energy density

i.e., specific

optical absorption

, the PA technique has the advantage of

directly measuring the absorption spectra

in vivo

, allowing

better tissue identification and providing functional

information.

Thus PA imaging is complementary with

other high resolution optical imaging modalities such as con-

focal microscopy, two-photon microscopy, and optical coher-

ence tomography, which can provide

in vivo

imaging within

the optical transport mean free path

1mm

of biological

tissues.

Previously, an optical-resolution confocal photoacoustic

microscope

OR-PAM

was described, with a lateral reso-

lution of

, axial resolution of

, and imaging depth

greater than

0.7 mm

OR-PAM is a good tool to image blood

vessels at the capillary level. High spatial resolution of OR-

PAM can resolve capillaries smaller than

in diameter

using hemoglobin within red blood cells

RBCs

as an en-

dogenic contrast agent.

However, RBC flow in capillaries is discontinuous and

changes greatly over time.

–

Because RBCs are the only

noticeable optical absorbers in capillaries, it is highly likely

that no absorber is present in a particular voxel during the

laser pulse, which results in discontinuous capillaries in a

RBC-based PA image. To acquire a complete capillary image

and gain information about the capillary’s functional state,

the use of an exogenic contrast agent is compelling.

In this work we use Evans blue

dye for this purpose.

EB has strong absorption in visible and near-infrared light,

with a peak at

620 nm

. EB is nontoxic and is used in mea-

surement of blood volume,

lymph node location,

mi-

crovascular permeability,

blood-retinal barrier

breakdown,

capillary perfusion,

and blood plasma flow,

among other applications. In the blood stream, EB mainly

binds to serum albumin in a reversible manner, so it is uni-

formly distributed in the plasma, maximizing the chance to

get a complete capillary network image. Under normal condi-

tions, the EB-albumin

EBA

complex is confined to blood

vessels, while the free dye more readily diffuses out into ex-

travascular tissue. The diffused dye is bound to the surround-

1083-3668/2009/14

Address all correspondence to: Lihong Wang, Dr., Optical Imaging Laboratory,

Department of Biomedical Engineering, Washington University in St. Louis, One

Brookings Dr., St. Louis, MO 63130. Tel: 314-935-6152; Fax: 314-935-7448;

E-mail: lhwang@seas.wustl.edu

Journal of Biomedical Optics 14

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ing tissue proteins, and finally cleared out by either metabo-

lism or excretion.

–

This work seeks to achieve complete capillary network

imaging, to quantitatively investigate the dynamic diffusion of

EB out of the circulation system, and to elucidate the dynamic

clearance of EBA.

2 Materials and Methods

2.1

Optical-Resolution Confocal Photoacoustic

Microscope System

The OR-PAM system used in this work has been described in

detail previously.

Briefly, a Nd:YLF laser

INNOSLAB,

523 nm

, Edgewave, Aachen, Germany

pumped a dye laser

CBR-D, Sirah, Karst, Germany

used as the irradiation

source. To cover the wavelength requirement for both RBCs

and EB, laser dye Pyrro597

peak at

582 nm

, tuning range

566 to 611 nm

was employed. The

7-ns

laser pulse was

passed through a

25-

-diam pinhole and focused by a mi-

croscope objective lens

Olympus

NA=0.1

. The laser

energy after the objective lens was

100 nJ

. Ultrasonic de-

tection was achieved through a spherically focused ultrasonic

transducer

V2012-BC, Panametrics-NDT, Olympus, focal

spot size

with a center frequency of

75 MHz

, and a

roundtrip

6-dB

bandwidth of 80%, placed confocally with the

optical objective. The PA signal detected by the ultrasonic

transducer was amplified, digitized, and saved. The laser

power was monitored by a reference channel to compensate

for laser power instability. A volumetric image was generated

by recording the time-resolved PA signal

A-line

at each

horizontal location of the 2-D raster scan.

2.2

Animal Preparation

The ears of adult,

6- to 8-week

-old nude mice

Hsd: Athymic

Nude-Foxl

Harlan Company, Indianapolis, Indiana, body

weight

20 g

were used for all

in vivo

experiments here,

because their small thickness allowed us to verify some of the

PA results by using a standard bright field optical microscope.

The nude mouse ear model has a well-developed vasculature

and has been widely used to study tumor angiogenesis and

other microvascular diseases.

Before data acquisition, the

animal was anesthetized by an intraperitoneal injection of

85% ketamine and 15% xylazine

100-

/

body weight

During data acquisition, the animal was placed on a warming

pad

37 °C

, and its head was held steady with a dental/hard

palate fixture. The animal was kept still by using a breathing

anesthesia system

E-Z Anesthesia, Euthanex, Palmer, Penn-

sylvania

. After the experiment, the animal recovered natu-

rally and was returned to its cage. All experimental animal

procedures were carried out in conformity with the laboratory

animal protocol approved by the Animal Studies Committee

of the School of Medicine at Washington University in Saint

Louis.

2.3

Mode of Injection of Evans Blue

A 6 or 3% EB w/v solution

Sigma, Saint Louis, Missouri

was prepared by dilution of the dye in phosphate-buffered

saline

PBS, pH 7.5

. Before injection, the solution was fil-

tered through a

filter. An intravenous injection of EB

was made to either of the dorsal veins of the tail. The injection

lasted for about

10 to 20 s

2.4

Spatially Continuous Capillary Imaging Using

Evans Blue as a Contrast Agent

Two irradiation wavelengths 570 and

610 nm

were chosen for

RBC imaging and EB imaging, respectively. An area of

2mm

was chosen as the field of interest

FOI

near the

margin of the nude mouse ear, where the capillary density was

higher. Before the dye injection, control images were acquired

with a scanning step size of

2.5

at 570 and

610 nm

. The

total scanning time for a complete volumetric dataset was

30 min

for each wavelength. To get sufficient imaging con-

trast and sensitivity of the capillaries, a relatively high con-

centration of EB in the blood plasma should be reached. Here

0.2 mL

of 6% EB solution was injected in a nude mouse

body weight

20 g

. The total blood volume of the mouse

was about

1.2 mL

Thus the concentration of EB in the

blood stream was

, corresponding to an absorption co-

efficient of

1000 cm

−1

610 nm

which is

times

higher than that of blood

50 cm

−1

. Two PA images at

610 nm

were acquired, one immediately after the dye injec-

tion and the other

30 min

later. Transmission optical micro-

scopic images at

magnification were acquired before and

after injection.

2.5

Dynamics of Evans Blue Diffusion Out of the

Blood Stream

EB is removed from the vascular system principally by dif-

fusing into extravascular tissue. At high dye concentrations, in

the first few hours, it is mainly the free EB rather than the

EB-albumin complex that diffuses out. The fixation of the free

EB molecules by tissue proteins causes more dye to leave the

blood to maintain chemical equilibrium, until the tissue pro-

teins become saturated.

–

To better understand the diffu-

sion dynamics, we monitored the dye diffusion over time and

quantified the diffused dye volume in the tissue. Here, a

smaller area of

1mm

was imaged near the margin of the

nude mouse ear, so more datasets could be acquired over time

due to the shorter scanning time of

10 min

. Control images

570 nm 610 nm

were acquired before dye injection. After

0.1 mL

of 6% EB solution was injected, the dye molar con-

centration in the plasma was

0.52 mM

, which was a high

concentration compared with the combination capability of

albumin.

Right after injection, serial images at

610 nm

were

acquired every

20 min

until the dye diffusion was observed to

have reached saturation.

2.6

Dynamics of the Evans Blue Albumin Clearance

At low dye concentrations, EB exists almost exclusively in

the form of EBA.

EB permeates wherever albumin is

present. Therefore, the clearance dynamics of the EBA may

be used to estimate the albumin metabolic rate in tissue.

better understand the clearance dynamics, in our work the

EBA volume in the tissue was monitored by PA imaging. An

imaging area of

1mm

was chosen on the nude mouse ear,

and control images at 570 and

610 nm

were acquired before

dye injection. Then

0.05 mL

of 3% EB solution was injected

Yao et al.: Evans blue dye-enhanced capillary-resolution photoacoustic microscopy

in vivo

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September/October 2009

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via the tail vein. Serial images were acquired at

610 nm

every

one or two days, until EBA had almost completely cleared

out.

2.7

Quantitative Data Processing

All the data processing in this work was based on the volu-

metric datasets. A typical 3-D data cube in this work consists

200

400

voxels for a

1-mm

scanning area, or

200

800

voxels for a

2-mm

scanning area. Each

voxel equals a physical size of

7.5

2.5

. Hilbert

transformation was performed along each A-line to extract the

amplitude information. For visualization, a maximum ampli-

tude projection

MAP

image was obtained by projecting the

maximum value of each A-line onto the transverse scanning

plane. The EB or EBA volume was estimated by count-

ing the voxels.

3 Results

3.1

Spatially Continuous Capillary Imaging Using

Evans Blue as a Contrast Agent

In this study, EB solution

6%,

0.2 mL

was injected into the

blood circulation system of the nude mouse. Before dye in-

jection, a PA image was acquired at

570 nm

Fig.

. He-

moglobin has strong absorption at this wavelength, which

provided high imaging contrast and a high signal-to-noise ra-

tio

40 dB

. Veins and arteries larger than

in diam-

eter contained a higher area density of RBCs and appeared

uniformly bright. However, smaller capillaries, containing a

single of RBCs, looked discontinuous and fragmentary

see

the arrows in Fig.

. As a control, another image was

acquired at

610 nm

Fig.

. The signal was very weak due

to the low hemoglobin absorption at

610 nm

, which was only

one-twentieth of that at

570 nm

. Right after the dye injection,

the microvascular network appeared continuous, as shown in

Fig.

. Dense capillaries could be observed, as indicated by

arrows. All the capillaries that appear “broken” in Fig.

became smooth and continuous

Video 1

. Moreover, the cap-

illary branching points that were invisible in Fig.

could

be clearly distinguished. The blood vessels in Fig.

appear

somewhat thicker than those in Fig.

, which was possibly

because the plasma volume was larger than the RBC volume.

The discernable blood vessel volume in the plasma-based im-

age appeared to be more than 50% greater than that disclosed

in the RBC-based image. The image in Fig.

was acquired

610 nm 30 min

after dye injection. It shows that a consid-

erable amount of EB had diffused out of the blood vessels into

the surrounding tissue but did not diffuse into the sebaceous

glands, which appear as brown patches in the transmission

microscopic images

see arrows in Figs.

–

3.2

Dynamics of Evans Blue Diffusion Out of the

Blood Stream

As before, control images at 570 and

610 nm

were acquired

before dye injection

Figs.

and

. The whole mi-

crovascular network within the field of view showed up with

denser and more continuous capillaries right after the

0.1 mL

Fig. 1

EB enhanced photoacoustic imaging of mouse ear microves-

sels. PA microvascular image before dye injection acquired at

570 nm and at

610 nm. Arrows in

point to the fragmentary

capillaries.

PA image acquired at 610 nm right after EB

6%,

0.2 mL

injection via tail vein. Arrows in

point to the continuous

capillaries.

PA image acquired at 610 nm acquired 30 min after

injection. Transmission microscopic images of the same area

be-

fore and

after injection. Arrows in

,and

point to seba-

ceous glands. All the photoacoustic images were scaled to the same

level of PA signal.

Video 1

A volumetric visualization of the images before dye injection

at 570 nm and after dye injection at 610 nm

MOV, 0.9 MB

URL:

http://dx.doi.org/10.1117/1.3251044.1

Fig. 2

Dynamics of EB diffusion out of the blood stream into sur-

rounding tissue. PA images acquired before EB injection at

570 nm

and at

610 nm

through

PA images acquired at 610 nm after

6%, 0.1 mL

injection at different times.

Partial volume of EB

diffused into surrounding tissue. An exponential recovery model was

used to fit the experiment data. All the photoacoustic images were

scaled to the same level.

Yao et al.: Evans blue dye-enhanced capillary-resolution photoacoustic microscopy

in vivo

Journal of Biomedical Optics

September/October 2009

Vol. 14

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