Functional transcranial brain imaging by optical-resolution photoacoustic microscopy

Functional transcranial

brain imaging by

optical-resolution

photoacoustic microscopy

Song Hu, Konstantin Maslov, Vassiliy Tsytsarev,

and Lihong V. Wang

Washington University in St. Louis, Department of

Biomedical Engineering, Optical Imaging Laboratory, One

Brookings Drive, St. Louis, Missouri 63130-4899

Abstract.

Optical-resolution photoacoustic microscopy

OR-PAM

is applied to functional brain imaging in living

mice. A near-diffraction-limited bright-field optical illumi-

nation is employed to achieve micrometer lateral reso-

lution, and a dual-wavelength measurement is utilized to

extract the blood oxygenation information. The variation

in hemoglobin oxygen saturation

along vascular

branching has been imaged in a precapillary arteriolar tree

and a postcapillary venular tree, respectively. To the best

of our knowledge, this is the first report on

in vivo

volu-

metric imaging of brain microvascular morphology and

oxygenation down to single capillaries through intact

mouse skulls. It is anticipated that:

chronic imaging

enabled by this minimally invasive procedure will ad-

vance the study of cortical plasticity and neurological dis-

eases;

revealing the neuroactivity-dependent changes

in hemoglobin concentration and oxygenation will facili-

tate the understanding of neurovascular coupling at the

capillary level; and

iii

combining functional OR-PAM

and high-resolution blood flowmetry will have the poten-

tial to explore cellular pathways of brain energy

metabolism.

2009 Society of Photo-Optical Instrumentation Engineers.

DOI: 10.1117/1.3194136

Keywords: optical-resolution photoacoustic microscopy; transcranial

brain imaging; label-free; capillary; hemoglobin oxygen saturation;

vascular branching; cortical plasticity; neurovascular coupling

Paper 09119LR received Apr. 1, 2009; revised manuscript received

Jun. 9, 2009; accepted for publication Jun. 17, 2009; published online

Aug. 7, 2009.

Advances in brain imaging facilitate the understanding of

cognitive phenomena and neurological diseases. However,

high-resolution brain imaging through intact animal skulls re-

mains challenging for pure optical modalities because the op-

tical scattering and absorption of the skull degrade the imag-

ing resolution and signal-to-noise ratio

SNR

. Photoacoustic

imaging, combining light and ultrasound in a single hybrid

technology, suggests a potential solution. Using acoustic-

resolution photoacoustic microscopy

AR-PAM

, Stein et al.

recently demonstrated mouse brain imaging through both in-

tact scalp and skull.

This noninvasive feature is highly desir-

able for functional or chronic studies; however, with its cur-

rent spatial resolution

lateral resolution:

; axial

resolution:

, capillaries are not resolvable. To fill the

gap, Maslov et al. developed optical-resolution photoacoustic

microscopy

OR-PAM

capable of imaging single capillaries

in vivo

The lateral resolution of OR-PAM matches the size

of a single red blood cell

RBC

, and its sensitivity enables

single RBC detection.

Here, we report on the first demonstration of OR-PAM for

functional brain microvascular imaging down to single capil-

laries through intact mouse skulls. The minimally invasive

feature is favorable for chronic study of cortical plasticity.

Moreover, because neuronal activity is widely assumed to

spatially correlate most closely to the capillary bed response,

improving localization of signals down to the capillary level

will enable functional brain mapping at micrometer reso-

lution.

Before functional brain imaging, a Swiss Webster mouse

Hsd:ND4, Harlan Co.,

25–30 g

was anesthetized by intra-

peritoneally administering a dose of

87 mg

/

ketamine and

13 mg

/

xylazine and transferred to a stereotaxic imaging

stage. The scalp of the mouse was surgically removed, and the

exposed skull was cleaned with 0.9% sodium chloride irriga-

tion solution right before imaging. Ultrasonic gel was used for

ultrasound coupling and maintaining skull hydration.

Throughout the experiment, the animal was supplied with

breathing-grade air and maintained under anesthesia using va-

porized isoflurane

1.0–1.5% isoflurane with an airflow rate of

/

min

. The body temperature of the animal was main-

tained at

37 °C

by a temperature-controlled heating pad. At

the end of the experiment protocol, the animal was euthana-

tized by an intraperitoneal administration of pentobarbital at a

1083-3668/2009/14

Address all correspondence to: Lihong V. Wang, Tel:

314

935-6152; Fax:

314

935-7448; E-mail: lhwang@biomed.wustl.edu

Fig. 1

In vivo

functional OR-PAM imaging of mouse brain microvas-

culature through an intact skull.

MAP image acquired at 570 nm;

MAP image acquired at 578 nm;

vessel-by-vessel sO

map-

ping;

a venular tree with branching orders

boxed by blue dashed

lines in

;

an arteriolar tree with branching orders

boxed by red

dashed lines in

. The calculated sO

values are shown in the color

bar. PA: photoacoustic signal amplitude. The scale bar applies to

,and

Color online only.

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dosage of

100 mg

/

. The detailed system description can be

found in our published papers.

In photoacoustic measurements of hemoglobin concentra-

tion and oxygenation, we assume that, in the visible spectral

range, oxyhemoglobin

HbO

and deoxyhemoglobin

HbR

are the dominant absorbing compounds in blood.

Thus, a

dual-wavelength measurement is adequate to image

though using more wavelengths is expected to yield more

accurate results.

The two wavelengths chosen here are 570

and

578 nm

, where the absorption contrast between blood and

background brain tissues are high enough to enable satisfac-

tory imaging quality.

According to the published absorption

spectra of rat hemoglobin,

570 nm

is an isosbestic point, at

which

HbO

and HbR have the same molar extinction coef-

ficients. Thus, the photoacoustic signal acquired at this wave-

length reflects the total hemoglobin concentration

HbT

, and

578 nm

is an

HbO

-absorption-dominant wavelength, which

helps differentiate the two types of hemoglobin. Because

blood oxygenation is highly correlated with local metabolism,

the

value is generally time variant. To minimize measure-

ment error due to the possible temporal fluctuation in blood

oxygenation, we implemented a wavelength autotuning pro-

gram to control the dye laser, and imaged the same cross-

sectional scan

B-scan

for each of the wavelengths before

moving to the next B-scan. The region of interest

ROI

was

scanned with a step size of

2.5

. The dual-wavelength

measurement took

20 min

, which was mostly limited by the

wavelength tuning speed of the dye laser.

Figures

and

are maximum amplitude projection

MAP

images of the mouse brain microvasculature under

systemic normoxia at the optical wavelengths of 570 and

578 nm

, respectively. Because

570 nm

is an isosbestic point,

Fig.

maps the HbT, regardless of the blood oxygenation.

Microvessels labeled with CL in Fig.

appear to be single

capillaries with diameters of

5–10

. A corresponding

volumetric rendering is shown in

Video 1

. At the conclusion

of the dual-wavelength measurement, the concentrations of

HbR and

HbO

, as well as the

values, were calculated

based on the model described in previously published work.

As shown in Fig.

, different

levels are visualized with

pseudocolors ranging from blue to red, while the HbT mea-

sured at the isosbestic point

570 nm

is represented by pixel

brightness. According to known physiology, the red vessels

values of

90%

are believed to be arterioles, whereas

the green ones

values as low as 60–70%

are most likely

to be venules. However, in the microcirculation, there is no

clear cutoff in

value between arterioles and venules.

The

blood oxygenation is closely associated with the microvascu-

lar branching order

and the local metabolic activity of the

tissue.

To demonstrate the oxygen gradients in the brain mi-

crocirculation, we selectively analyzed a postcapillary venular

tree

Fig.

and a precapillary arteriolar tree

Fig.

highlighted in Fig.

by the blue and red dashed boxes,

respectively. The vessel diameters and the corresponding

values in different branching orders are listed in Tables

and

. A negative correlation between the branching order and its

mean

value is observed in both the arteriolar and venular

trees with a linear regression analysis

arteriolar tree:

=0.98

=0.01;

venular tree:

=0.75

=0.14

, as shown in

Table

The vessel diameters and the sO

values in different branching orders in a venular tree. Values are in mean±standard deviation format.

Branching

order

Vessel ID

Diameter

57±2

48±5

42±3

35±2

30±2

41±2

24±3

20±2

19±3

83±3

85±2

86±3

83±3

78±2

75±4

74±5

76±4

Table

The vessel diameters and the sO

values in different branching orders in an arteriolar tree. Values are in mean±standard deviation format.

Branching

order

Vessel ID

Diameter

59±4

48±3

43±4

52±2

44±4

32±2

51±2

42±4

23±3

24±2

93±2

91±3

89±2

88±4

87±3

79±3

85±2

81±3

Video 1.

In vivo

volumetric visualization of the mouse brain mi-

crovasculature through an intact skull by OR-PAM

QuickTime 4 MB

URL: http://dx.doi.org/10.1117/1.3194136.1

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Fig.

. Our results suggest that:

the blood oxygenation

level is higher in the arteriolar tree than in the venular tree;

capillaries are not the only oxygen exchange site in the

microcirculation because the

decreases significantly with

vascular branching in the precapillary arteriolar tree

12%

from order 1 to 4

; and

iii

a diffusional shunt is present

between arterioles and venules to elevate the oxygen level in

“large” venules because the

increases noticeably with

blood confluence in the postcapillary venular tree

from

order 1 to 4

. Our observation is in agreement with the pub-

lished work.

–

According to a previous study,

the wavelength-dependent

optical attenuation due to the intact skull affects the measure-

ment accuracy. Here, this wavelength dependence was esti-

mated by measuring the photoacoustic signals of a black poly-

ethylene film attached beneath the skull of a freshly sacrificed

mouse of the same type at the two operating wavelengths,

respectively. The mouse skull was submerged in 0.9% sodium

chloride irrigation solution to keep hydration. Because the

black polyethylene film can be considered as a neutral ab-

sorber in the visible spectral range, the wavelength depen-

dence in the photoacoustic signals is expected to be predomi-

nantly from the mouse skull. If we consider the skull to be

homogeneous within the ROI,

then such dependence can be

simply compensated for by applying different calibration fac-

tors to the measured photoacoustic signals at different optical

wavelengths. However, the wavelength-dependent optical

scattering and absorption of brain tissues are difficult to com-

pensate for due to heterogeneous tissue structure and compo-

sition.

In summary, OR-PAM has been applied to mouse brain

imaging through intact skulls with capillary-level spatial res-

olution. Functional information of HbT and

within single

microvessels was imaged simultaneously using a dual-

wavelength measurement. The ability to extract brain oxygen

saturation information on a single-capillary basis with mini-

mal invasiveness makes OR-PAM a potential tool for high-

resolution functional brain mapping, quantitative analysis of

brain energy metabolism, and chronic studies of cortical plas-

ticity and neurological diseases. It is worth noting that the

maximum imaging depth of OR-PAM, estimated from the sur-

face of the intact skull, is

500–600

when the system is

operated at the Q-band of the hemoglobin absorption spec-

trum

optical wavelength:

560 nm

. This penetration is

slightly less than the typical imaging depth of two-photon

microscopy

TPM

600–800

through a skull window

with near-infrared

NIR

excitation,

where no bone is in the

optical path. NIR light sustains less brain tissue absorption

and scattering than visible light. However, owing to the qua-

dratic intensity dependence in two-photon generation and lin-

ear dependence in OR-PAM, the light scattering and absorp-

tion within the intact skull as well as the surface scattering at

the skull-brain interface decreases the fluorescence signal in

TPM much faster than the photoacoustic signal in OR-PAM.

As a result, TPM has not demonstrated transcranial imaging.

By utilizing NIR operation and compromising the lateral res-

olution, doppler optical coherence tomography can extend the

imaging depth to

1.5 mm

To enhance the penetration of

OR-PAM, system SNR or imaging contrast needs to be fur-

ther improved.

Acknowledgments

The authors appreciate Prof. James Ballard’s close reading of

the paper. This work was sponsored by National Institutes of

Health Grants No. R01 EB000712, No. R01 NS46214, No.

R01 EB008085, and No. U54 CA136398. L.W. has a financial

interest in Endra, Inc., which, however, did not support this

work.

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Fig. 2

The sO

value versus the vascular branching order in a precap-

illary arteriolar tree and a postcapillary venular tree. The square and

round markers represent the mean sO

values in each branching or-

der, and the error bars stand for the standard deviations of the mean

values.

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