Microvascular autoregulation is an intrinsic ability of vascular beds to compensate for the fluctuation in blood flow and tissue oxygen delivery. This function is crucial to maintaining the local metabolic activity. Here, using optical-resolution photoacoustic microscopy (OR-PAM), we clearly observed vasomotion and vasodilation in the intact mouse microcirculation in vivo in response to the changes in physiological state. Our results show that a significant lowfrequency vasomotion can be seen under hyperoxia but not hypoxia. Moreover, significant vasodilation is observed when the animal status is switched from hyperoxia to hypoxia. Our data show that arterioles have more pronounced vasodilation than venules

Hu, Song

Maslov, Konstantin I.

Wang, Lihong V.

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In vivo noninvasive monitoring of
microhemodynamics using optical-
resolution photoacoustic microscopy
Song  Hu, Konstantin I. Maslov, Lihong V. Wang
Song  Hu, Konstantin I. Maslov, Lihong V. Wang, "In vivo noninvasive
monitoring of microhemodynamics using optical-resolution photoacoustic
microscopy," Proc. SPIE 7177, Photons Plus Ultrasound: Imaging and
Sensing 2009, 71770H (24 February 2009); doi: 10.1117/12.809297
Event: SPIE BiOS, 2009, San Jose, California, United States
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In Vivo Noninvasive Monitoring of Microhemodynamics Using 
Optical-Resolution Photoacoustic Microscopy 
 
Song Hu, Konstantin I. Maslov, and Lihong V. Wang 
 
Optical Imaging Laboratory, Department of Biomedical Engineering,  
Washington University in St. Louis, St. Louis, Missouri, USA 63130  
ABSTRACT 
Microvascular autoregulation is an intrinsic ability of vascular beds to compensate for the fluctuation in blood flow and 
tissue oxygen delivery. This function is crucial to maintaining the local metabolic activity. Here, using optical-resolution 
photoacoustic microscopy (OR-PAM), we clearly observed vasomotion and vasodilation in the intact mouse 
microcirculation in vivo in response to the changes in physiological state. Our results show that a significant low-
frequency vasomotion can be seen under hyperoxia but not hypoxia. Moreover, significant vasodilation is observed 
when the animal status is switched from hyperoxia to hypoxia. Our data show that arterioles have more pronounced 
vasodilation than venules.  
   
Keywords: Optical-resolution photoacoustic microscopy, microvascular autoregulation, vasomotion, vasodilation. 
 
1. INTRODUCTION 
 
Microcirculation is the terminal functional unit of the cardiovascular system, carrying out the exchange of gas and 
nutrients between blood and tissue. In vivo noninvasive microvascular imaging is of great physiological and clinical 
importance. However, until now, there has not been a technique that could provide comprehensive and accurate 
information of the intact microcirculation.  
 
To fill this gap, we have developed optical-resolution photoacoustic microscopy (OR-PAM)1, 2. Benefiting from the 
physiologically specific endogenous optical absorption contrast, OR-PAM enables in vivo noninvasive contrast-agent-
free three-dimensional microvascular imaging down to the capillary level. 
 
In this work, we monitored vasomotion and vasodilation—two important microhemodynamic activities in tissue oxygen 
regulation—in the intact microcirculation in small animals in vivo. The good agreements between our observations and 
the previous invasive studies validate the feasibility of OR-PAM. The noninvasiveness and label-free feature of OR-
PAM enables a more accurate and objective view on the microcirculation. 
 
2. METHODS AND MATERIALS 
 
2.1 OR-PAM with diffraction-limited optical focusing 
As shown in Fig. 1(A), in our OR-PAM system, a wavelength-tunable laser set, consisting of a dye laser an Nd:YLF 
pump laser, is used as the excitation source. Laser beam coming out of the dye laser is attenuated and spatially filtered 
by a pinhole. The reshaped laser beam is then focused into a diffraction-limited optical focal spot by an objective lens. 
Ultrasonic focusing is achieved through a plano-concave attached under the right-angle prism. The optical objective lens 
and the ultrasonic transducer are configured coaxially and confocally.  
 
Photons Plus Ultrasound: Imaging and Sensing 2009, edited by Alexander A. Oraevsky, Lihong V. Wang,
Proc. of SPIE Vol. 7177, 71770H · © 2009 SPIE · CCC code: 1605-7422/09/$18 · doi: 10.1117/12.809297
Proc. of SPIE Vol. 7177  71770H-1
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In this acoustic-optical confocal configuration, the lateral resolution is determined by the product of the two point-
spread-functions of optical illumination and acoustic detection. Here, we use diffraction-limited optical focusing to 
achieve the capillary-level lateral resolution. As shown in Fig. 1(B), within the 27-µm-diameter acoustic focus, only a 5-
µm-diameter spot is optically excited, so the detected photoacoustic signal is exclusively generated within the optical 
focus. Thereby, the lateral resolution of photoacoustic microscopy can be easily promoted from 50 µm3-5 to 5 µm or 
even higher. 
 
 
B
Condenser lens
Pinhole
Objective lensCorrection lens
Water tank
Right-angle prism
Silicone oil layeri
Animal
Ultrasonic 
transducer
Scanner
Wavelength 
tunable laser set
Membrane
Acoustic 
lens
Laser beam
Dual foci
A
 
Figure 1. (A) Schematic of the OR-PAM system1, 2. (B) A blow up sketch of the diffraction-limited optical illumination and the 
acoustic-optical confocal configuration. 
 
2.2 Animal preparation 
All experimental animal procedures were carried out in conformance with the laboratory animal protocol approved by 
the School of Medicine Animal Studies Committee of Washington University in St. Louis. 
 
Nude mice (Hsd:Athymic Nude-Foxn 1NU, Harlan Co.) were used. Before imaging, the hair on the ears was gently 
removed with human hair-removing lotion. A dose of 87 mg/kg Ketamine and 13 mg/kg Xylazine was administered 
intraperitoneally to anesthetize the animal, and anesthesia was maintained throughout the experiments using an 
isoflurane machine (1.0-1.5% vaporized isoflurane with an airflow rate of 1 liter/min). The body temperature of the 
animal was maintained at 37ºC with a temperature controlled heating pad. At the end of the experiment, the animals 
were killed by an intraperitoneal administration of pentobarbital at a dosage of 100 mg/kg. 
 
 
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3. RESULTS AND DISCUSSION 
 
3.1 In vivo microvascular imaging 
A 1mm-by-1mm region in a nude mouse ear was imaged in vivo noninvasively by OR-PAM at the isosbestic wavelength 
of 570 nm. The region of interest was scanned with a step size of 2.5 µm. The scanning time for a complete volumetric 
dataset was ~6 min. After data acquisition, the animal recovered naturally without observable laser damage.  
 
50 µm
O
pt
ic
al
 a
bs
or
pt
io
n
M
in
M
axA
B
C
 
 
Figure 2. Microvasculature in a nude mouse ear. (A) The maximum-amplitude-projection image acquired in vivo by OR-PAM; (B) 3D 
pseudocolor visualization of the microvasculature; (C) The photograph taken by a transmission-mode optical microscope. 
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3.2 In vivo monitoring of vasomotion and vasodilation in intact microcirculations 
Vasomotion is the oscillation of vascular tone or vascular diameter that can be generally seen in the cardiovascular 
system6. It is well known not to be a consequence of the heart beat, respiration, or neuronal input; however, its 
physiological role and its underlying mechanism remain elusive7. Vasodilation is better understood to be increase in 
vessel diameter. Both of them are important vessel activities in tissue oxygen regulation. 
 
To study vasomotion and vasodilation in the intact microcirculation, we randomly imaged a microvascular network in an 
ear of a living nude mouse, and then selected a representative B-scan crossing several arterioles and venules for 
monitoring purpose. To induce vasomotion and vasodilation in vivo, we changed the tissue oxygenation of the 
experimental mouse by alternating the inspired gas between pure oxygen and hypoxic gas (5% O2, 5% CO2 and 90% 
N2). We monitored the selected B-scan continuously for seventy minutes during the switching of physiological state, as 
shown in Fig. 3. Vasomotion under hyperoxia is clearly seen in the form of the oscillation of the vessel cross sections; 
vasodilation under hypoxia is also clearly observed as the expansion of the vessel diameters. 
0 5 10 20 30 50 70 
V1
A2
A1B
-s
ca
n 
ax
is
20 µm
Hyperoxia Hypoxia
Time (min)
 
 
Figure 3. B-scan monitoring of the arteriolar and venular vasomotion and vasodilation in response to the changes in physiological 
state. A1 and A2 are two selected arterioles; V1 is the selected venule. 
 
 
Thanks to the high resolution and high sensitivity of OR-PAM measurements, we can study the vasodilation in a more 
quantitative way by estimating the vessel diameters using the full-width-at-half-maximum values of the vessel cross 
sectional signals. To eliminate the vessel diameter oscillation due to vasomotion, we employed 60-point-moving-
averaging. The smoothed time courses of the arteriolar and venular diameter changes in response to the switching of the 
physiological state are shown in Fig. 4. It clearly shows that arterioles A1 and A2 have a more significant vasodilation 
(80%-100%) than venule V1 (30%-35%). 
 
 
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Ve
ss
el
 d
ia
m
et
er
 (µ
m
)
Time (min)
0 10 20 30 40 50 60 700
10
20
30
40
50
60
70
 
 
A1
A2
V1
 
Figure 4. The arteriolar and venular vasodilation in response to hypoxia. 
 
The adequate temporal resolution of OR-PAM also permits quantifying the response time of vasodilation to hypoxia. As 
shown in Fig. 5, the response time is estimated to be 2-3 minutes. 
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0 10 20 30 40 50 60 70
5
10
15
20
25
 
 
Time (min)
Ve
ss
el
 d
ia
m
et
er
 (µ
m
)
10%
90%
~ 150 s
 
Figure 5. The response time of arteriolar vasodilation to hypoxia. 
 
4. CONCLUSIONS 
 
OR-PAM, tracing the physiologically specific absorption signatures of endogenous chromophores, is a valuable 
complement to the existing scattering- and fluorescence-contrast microscopic techniques. In this work, we successfully 
demonstrated in vivo monitoring of two important vessel activities—vasomotion and vasodilation—in the intact 
microcirculation by taking advantage of the endogenous optical absorption contrast of hemoglobin. Our results are in 
good agreement with the previous invasive studies. OR-PAM provides a more accurate and objective view on the 
microcirculation by avoiding the possible disturbance due to invasive procedures or exogenous contrast agents. 
 
 
ACKNOWLEDGEMENTS 
 
This work was sponsored by National Institutes of Health grants R01 EB000712, R01 NS46214 (Bioengineering 
Research Partnerships), R01 EB008085, and U54 CA136398 (Network for Translational Research). L.W. has a financial 
interest in Endra, Inc., which, however, did not support this work. 
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REFERENCES 
1. K. Maslov, H.F.Z., S. Hu, and L.V. Wang Optical-resolution confocal photoacoustic microscopy. Proc. SPIE 6856, 68561I 
(2008). 
2. Maslov, K., Zhang, H.F., Hu, S. & Wang, L.V. Optical-resolution photoacoustic microscopy for in vivo imaging of single 
capillaries. Opt Lett 33, 929-931 (2008). 
3. Maslov, K., Stoica, G. & Wang, L.V. In vivo dark-field reflection-mode photoacoustic microscopy. Opt Lett 30, 625-627 
(2005). 
4. Zhang, H.F., Maslov, K., Stoica, G. & Wang, L.V. Functional photoacoustic microscopy for high-resolution and 
noninvasive in vivo imaging. Nat Biotechnol 24, 848-851 (2006). 
5. Zhang, H.F., Maslov, K. & Wang, L.V. In vivo imaging of subcutaneous structures using functional photoacoustic 
microscopy. Nat Protoc 2, 797-804 (2007). 
6. Aalkaer, C. & Nilsson, H. Vasomotion: cellular background for the oscillator and for the synchronization of smooth muscle 
cells. British journal of pharmacology 144, 605-616 (2005). 
7. Nilsson, H. & Aalkjaer, C. Vasomotion: mechanisms and physiological importance. Molecular interventions 3, 79-89, 51 
(2003). 
 
 
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In vivo noninvasive monitoring of microhemodynamics using optical-resolution photoacoustic microscopy

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In vivo noninvasive monitoring of microhemodynamics using optical-resolution photoacoustic microscopy

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