Optical-resolution photoacoustic microscopy has demonstrated utility in imaging and characterizing microvasculature networks in vivo. This work presents a novel imaging system that integrates optical-resolution photoacoustic microscopy and fluorescence confocal microscopy for simultaneous photoacoustic and fluorescence imaging. The integrated system can acquire intrinsically registered photoacoustic and fluorescence images in a single scan. Capable of providing micrometer scale microscopic imaging of both optical absorption and fluorescence contrasts, the system demonstrates in vivo imaging of oxygen saturation and oxygen partial pressure in mouse ears

Hu, Song

Kim, Chulhong

Maslov, Konstantin

Wang, Lihong V.

Wang, Yu

PubMed

We have developed a dual-modality imaging system by integrating optical-resolution photoacoustic microscopy and fluorescence confocal microscopy to provide optical absorption and fluorescence contrasts simultaneously. By sharing the same laser source and objective lens, intrinsically registered photoacoustic and fluorescence images are acquired in a single scan. The micrometer resolution allows imaging of both blood and lymphatic vessels down to the capillary level. Simultaneous photoacoustic angiography and fluorescence lymphangiography were demonstrated, presenting more information to study tumor angiogenesis, vasculature, and microenvironments in vivo

Caltech Authors - Main

Integrated Photoacoustic and Fluorescence Confocal Microscopy
Yu Wang,
Optical Imaging Laboratory, Department of Biomedical Engineering, Washington University in St.
Louis, Campus Box 1097, One Brookings Drive, St. Louis, MO 63130-4899, USA
Konstantin Maslov,
Optical Imaging Laboratory, Department of Biomedical Engineering, Washington University in St.
Louis, Campus Box 1097, One Brookings Drive, St. Louis, MO 63130-4899, USA
Chulhong Kim,
Optical Imaging Laboratory, Department of Biomedical Engineering, Washington University in St.
Louis, Campus Box 1097, One Brookings Drive, St. Louis, MO 63130-4899, USA
Song Hu, and
Optical Imaging Laboratory, Department of Biomedical Engineering, Washington University in St.
Louis, Campus Box 1097, One Brookings Drive, St. Louis, MO 63130-4899, USA
Lihong V. Wang [Fellow, IEEE]
The Optical Imaging Laboratory, Department of Biomedical Engineering, Washington University in
St. Louis, Campus Box 1097, One Brookings Drive, St. Louis, MO 63130-4899, USA
Lihong V. Wang: lhwang@biomed.wustl.edu
Abstract
We have developed a dual-modality imaging system by integrating optical-resolution photoacoustic
microscopy and fluorescence confocal microscopy to provide optical absorption and fluorescence
contrasts simultaneously. By sharing the same laser source and objective lens, intrinsically registered
photoacoustic and fluorescence images are acquired in a single scan. The micrometer resolution
allows imaging of both blood and lymphatic vessels down to the capillary level. Simultaneous
photoacoustic angiography and fluorescence lymphangiography were demonstrated, presenting
more information about tumor angiogenesis, vasculature, and microenvironments in vivo.
Index Terms
Photoacoustic microscopy; Fluorescence confocal microscopy; Multimodal imaging; Angiography;
Lymphangiography
I. Introduction
Multimodal imaging provides complementary anatomical and functional contrasts of
biological tissue. Predominantly sensitive to optical absorption contrast, photoacoustic
microscopy (PAM) can noninvasively image both hemoglobin concentration and oxygen
saturation in single blood vessels using endogenous contrast [1], [2]. Such functional contrasts
have been co-registered with anatomical contrasts by combining photoacoustic (PA) imaging
with other imaging modalities, such as optical coherence tomography (OCT) [3], [4],
backscattering confocal microscopy (BCM) [5], and ultrasonic imaging (USI) [6], [7]. While
Correspondence to: Lihong V. Wang, lhwang@biomed.wustl.edu.
NIH Public Access
Author Manuscript
IEEE Trans Biomed Eng. Author manuscript; available in PMC 2010 November 3.
Published in final edited form as:
IEEE Trans Biomed Eng. 2010 October ; 57(10): 2576–2578. doi:10.1109/TBME.2010.2059026.
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OCT and BCM image optical scattering, USI senses acoustic scattering. Anatomical images
based on such scattering contrasts provide rich context for the functional PA images. Here, we
report a hybrid PA and fluorescence confocal microscopy (PA-FCM). Fluorescence confocal
microscopy (FCM) has already been applied in a broad variety of biological studies for imaging
fluorophores. We believe that our new hybrid imaging system can offer more comprehensive
insights into physiology through co-registered microscopic images of optical absorption and
fluorescence contrasts.
To demonstrate the PA-FCM system, we imaged blood vessels and draining lymphatic vessels
in vivo simultaneously. In vivo angiography and lymphangiography are invaluable in
microvascular-related physiological and pathophysiological research [8], [9]. Cancer
hallmarks include hypoxia, angiogenesis, and lymphangiogenesis [10]–[12]. Moreover, cancer
metastases can occur via blood or lymphatic vessels. Spectroscopic PA imaging has been
employed to study new blood vessel formation and hemoglobin oxygen saturation (sO2) in
tumors [13], [14]. Meanwhile, fluorescence microscopy has been widely used to image
lymphangiogenesis and lymphatic hyperplasia in mouse tumor models [15]. The combination
of PA angiography and fluorescence lymphangiography can provide more comprehensive
information for monitoring tumor growth and treatment in small animals.
II. Methods and materials
A. PA-FCM System
Figure 1 illustrates the experimental configuration of our PA-FCM system. The optical-
resolution PAM subsystem was described in detail previously [1]. Briefly speaking, a tunable
dye laser (Pyrromethene 597, Exciton, CBR-D, Sirah) pumped by a Nd:YLF laser
(INNOSLAB, Edgewave) serves as the irradiation source. The laser beam (pulse duration, 7
ns), after passing through the excitation pinhole (pinhole diameter, 25 μm), is focused into the
sample by an objective lens (NA, 0.2; magnification, 13.3). The laser pulse energy after the
objective lens is ∼100 nJ. The generated PA waves are reflected by an acoustic-optical beam
splitter to a 75-MHz ultrasonic transducer (V2022 BC, Olympus NDT). The FCM subsystem
shares the same objective lens. A dichroic mirror (DMLP605, Thorlabs) allows transmission
of fluorescence (above 605 nm) emitted from the sample. Two emission filters
(FF01-624/40-25, Semrock) further eliminate the reflected excitation light. The fluorescence
light passing through the detection pinhole (pinhole diameter, 50 μm) is collected by a
photomultiplier tube module (H6780-20, Hamamatsu). The diameter of the detection pinhole
was chosen by approximately matching the object-side pinhole diameter (detection pinhole
diameter divided by objective lens magnification = 3.8 μm) with one Airy unit (1.22λ/NA =
3.5 μm). This method is commonly used in FCM to improve signal-to-noise ratios, while
sufficiently rejecting light from off-focus plane [16], [17]. The amplified PA signal and
fluorescence signal are digitized by a data acquisition card (CS 14200, Gage Applied). Two-
dimensional raster scanning of the sample along the x-y transverse plane provides automatically
co-registered PA and fluorescence images. Dual-wavelength angiography and
lymphangiography images are captured by changing the laser wavelength after completing
each B-scan. For light fluence compensation for both PA and fluorescence images, the laser
energy is measured at both wavelengths by a photodiode before imaging.
B. Animal Preparation
We applied the multimodal system to image an ear of a nude mouse (Harlan, body weight
∼20 g) in vivo. To visualize the lymphatic network using PA-FCM, 5 μL of 20% rhodamine
B isothiocyanate (RITC)–dextran (R9379, Sigma; average molecular weight ∼70000) was
injected into the mouse ear tip using a 29-gauge needle. Imaging of blood and lymphatic vessels
started immediately after the dye injection and lasted for ∼40 min. To keep the animal
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motionless, a breathing anesthesia system (E-Z Anesthesia, Euthanex) ventilated a gas mixture
of 1% isoflurane and medical grade oxygen to the mouse during experiments. All experimental
animal procedures were carried out in conformity with the laboratory protocol approved by
the Animal Studies Committee of the School of Medicine at Washington University in St.
Louis.
III. Results
A. Spatial resolution of PA-FCM system
The spatial resolution of the PA-FCM system was measured experimentally. To quantify the
resolution of PAM, a surgical blade was used as the sample. Figure 2(a) shows the edge spread
function from the blade PAM image. The measured edge spread function was nonlinearly fitted
by an error function. The full width at half maximum (FWHM) of its derivative —defining the
lateral resolution—was estimated to be ∼3.9 μm. The axial resolution of PAM was measured
using a “shift-and-sum” method with a single A-line PA signal in Fig. 2(b) [18]. The estimated
PAM axial resolution was ∼17 μm, as determined by the 100-MHz bandwidth of the transducer.
We quantified the resolution of confocal microscopy by measuring reflected light from a
cracked silver mirror surface. Emission filters were removed for the resolution test. The edge
spread function of the confocal microscope, as plotted in Fig. 2(c), yielded a lateral resolution
of ∼3.9 μm. To measure the axial resolution of the confocal microscope, the mirror was
translated along the axial direction. Figure 2(d) plots the normalized reflected light intensity
with the mirror surface at various depths. The experimental data was then fitted by a (sinc)4
function [16], whose FWHM of ∼38 μm was used as the axial resolution of the confocal
microscope.
B. Vascular and Lymphatic Imaging
Anatomical and functional images of a nude mouse ear were acquired at dual wavelengths (570
and 593 nm). Figure 3(a) shows a structural PA image of vasculatures in the ear taken at an
optical wavelength of 570 nm. Because 570 nm is an isosbestic point of oxyhemoglobin (HbO2)
and deoxyhemoglobin (HbR) molar absorptivities, the PA signal amplitude measured at this
wavelength depends on the total hemoglobin (HbT) concentration but not the sO2. Aided by
the PA-FCM images acquired at 593 nm, the relative concentrations of HbO2 and HbR, and
subsequently sO2, can be calculated based on the molar extinction spectra of HbO2 and HbR
[9]. The PA sO2 mapping is shown in Fig. 3(b). Figure 3(c) shows the fluorescence images of
lymphatics obtained simultaneously with the PA image in Fig. 3(a). After injection of RITC-
dextran, lymphatic networks became visible in the FCM image. The mean ratio of the
fluorescence signals from the lymphatic network at the excitation wavelengths of 570 nm and
593 nm was 9.2, and the ratio measured with the fluorescence dye in vitro was 10.0, confirming
fluorescence emission from RITC-dextran. Figure 3(d) co-registers the mappings of the total
hemoglobin concentration and fluorescence lymphatics. Figure 3(e), the fused image of vessel-
by-vessel sO2 and fluorescent lymphatics, clearly depicts arterioles, venules, and draining
lymphatic vessels in the mouse ear. Because vasculature regulates the metabolic and
hemodynamic states of biological tissue and angiogenesis and lymphangiogenesis facilitate
the spread of cancer, visualizing both blood and lymphatic vessels enables more comprehensive
tracking of cancer metastasis.
IV. Conclusion
The experimental results here demonstrate the capability of the PA-FCM system in revealing
complementary PA and fluorescence contrasts. The applications of PA-FCM will not be limited
to the fusion of angiography and lymphangiography. Fluorescent molecular probes for tumor
detection can be used so that tumor location and angiogenesis can be visualized simultaneously.
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With voltage-sensitive dye, fluorescence imaging can map voltage changes of the cell
membrane potential [19]; while PAM can image the vasoconstriction and vasodilatation of
blood vessels [2]. Hence the PA-FCM system may be used to image neurovascular interactions
during brain electrical stimulation.
Acknowledgments
This work was sponsored in part by National Institutes of Health grants R01 EB000712, R01 EB008085, R01
CA113453901, U54 CA136398, and 5P60 DK02057933. L.W. has a financial interest in Microphotoacoustics, Inc.
and Endra, Inc., which, however, did not support this work.
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Fig. 1.
Schematic of the dual-modality PA-FCM system.
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Fig. 2.
Spatial resolutions of the dual-modality PA-FCM. (a) The lateral edge spread function of the
PAM. (b) The axial resolution of the PAM. (c) The lateral edge spread function of the confocal
microscope. (d) The axial resolution of the confocal microscope.
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Fig. 3.
Combined functional vascular and lymphatic images of a mouse ear in vivo. (a) PAM image
of microvasculature acquired at 570 nm. (b) PA sO2 mapping based on dual-wavelength (570
nm and 593 nm) measurements. (c) FCM image of the lymphatic system acquired at 570 nm
after the injection of 20% RITC-dextran. (d) Fused mapping of total hemoglobin concentration
and fluorescent lymphatics. (e) Fused mapping of sO2 and fluorescent lymphatics. (f) Optical
microscopic image acquired within the same region. FL: fluorescence. OM: optical
microscopy.
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Integrated Photoacoustic and Fluorescence Confocal Microscopy

PROCEEDINGS OF SPIE
SPIEDigitalLibrary.org/conference-proceedings-of-spie
Integrated photoacoustic and
fluorescence confocal microscopy
Yu  Wang, Konstantin  Maslov, Chulhong  Kim, Song  Hu,
Lihong V. Wang
Yu  Wang, Konstantin  Maslov, Chulhong  Kim, Song  Hu, Lihong V. Wang,
"Integrated photoacoustic and fluorescence confocal microscopy," Proc. SPIE
7899, Photons Plus Ultrasound: Imaging and Sensing 2011, 78990M (17
February 2011); doi: 10.1117/12.874888
Event: SPIE BiOS, 2011, San Francisco, California, United States
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Integrated photoacoustic and fluorescence confocal microscopy 
 
Yu Wang, Konstantin Maslov, Chulhong Kim, Song Hu, Lihong V. Wang* 
Department of Biomedical Engineering, Washington University in St. Louis, Campus Box 1097, 
One Brookings Drive, St. Louis, MO 63130 
ABSTRACT 
Optical-resolution photoacoustic microscopy has demonstrated utility in imaging and characterizing microvasculature 
networks in vivo. This work presents a novel imaging system that integrates optical-resolution photoacoustic microscopy 
and fluorescence confocal microscopy for simultaneous photoacoustic and fluorescence imaging. The integrated system 
can acquire intrinsically registered photoacoustic and fluorescence images in a single scan. Capable of providing 
micrometer scale microscopic imaging of both optical absorption and fluorescence contrasts, the system demonstrates in 
vivo imaging of oxygen saturation and oxygen partial pressure in mouse ears.  
Keywords: Photoacoustic microscopy, fluorescence confocal microscopy, oxygen saturation, oxygen partial pressure 
 
1. INTRODUCTION 
Optical resolution photoacoustic microscopy (PAM) is a noninvasive volumetric imaging method predominantly 
sensitive to optical absorption [1-4]. It is able to acquire micrometer resolution images in vivo using endogenous 
absorption contrast, such as hemoglobin and melanin. Noninvasively imaging of both hemoglobin concentration and 
oxygen saturation in single blood vessels using PAM has been demonstrated. Other common forms of optical imaging 
modalities, such as optical coherence tomography (OCT) and fluorescence confocal microscopy (FCM) are based on 
optical scattering or fluorescence contrasts, respectively. Structural and functional images based on these different 
imaging contrasts provide complementary information for biological studies.  
PAM and OCT have been combined previously for multimodal retina imaging [5]. It provides volumetric imaging of the 
retinal microvasculature using PAM and the retinal structure using OCT. Here, we developed a dual-modality imaging 
system which integrates photoacoustic microscopy and fluorescence confocal microscopy (PA-FCM). Co-registered 
images of optical absorption contrast and fluorescence contrast can be acquired simultaneously. By imaging fluorophore, 
FCM can provide not only rich context for PAM vasculature imaging, but also complementary functional information 
for more comprehensive insights into physiology.  
To demonstrate the PA-FCM system, we imaged the hemoglobin oxygen saturation (sO2) using PAM and oxygen partial 
pressure (pO2) using FCM in vivo in single blood vessels. In addition, PAM also mapped the total hemoglobin 
concentration (HbT) at the isosbestic wavelength. The multimodal microscopy system shows that the PAM system can 
be naturally integrated to FCM system which is one of the traditional optical image modalities. The natural combination 
of PAM and FCM is anticipated to encourages the commercialization of PAM imaging technique as a supplement of the 
mainstream optical imaging techniques for routine laboratory use. 
  
2. METHOD AND METERIALS 
2.1 PA-FCM system configuration 
The experimental configuration of our PA-FCM system is shown in figure 1 [6]. The system utilizes a wavelength-
tunable dye laser (CBD-D, Sirah) pumped by a Nd:YLF laser (INNOSLAB, Edgewave). Laser pulses from the dye laser 
(560 nm to 590 nm) are used to image hemoglobin absorption at multiple wavelengths. To excite the oxygen-sensitive 
phosphores, the 523 nm pump laser pulses are delivered into the microscope system. The laser pulse illuminating the 
sample has a pulse energy of ~ 100 nJ. Chromophores in the sample generate photoacoustic wave and/or fluorescence 
light after absorbing the excitation laser pulse. Photoacoustic waves and fluorescence lights are separated by an acoustic-
optical beam splitter. A 75 MHz ultrasonic transducer (V2022BC, Olympus NDT) is used to detect the PA waves. The 
Photons Plus Ultrasound: Imaging and Sensing 2011, edited by Alexander A. Oraevsky, Lihong V. Wang,
Proc. of SPIE Vol. 7899, 78990M · © 2011 SPIE · CCC code: 1605-7422/11/$18 · doi: 10.1117/12.874888
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fluorescence light after passing through the filter is collected by a photomultiplier tube module (H6780-20, Hamamatsu). 
The FCM subsystem use a 150 µm diameter detection pinhole to suppress the out-of-focus light. The photoacoustic and 
fluorescence signals are digitized and stored by a DAQ instrument (CS 14200, Gage Applied).  
 
 
Figure 1. Schematic of the integrated photoacoustic and fluorescence confocal microscopy system. 
  
 
2.2 PA-FCM system spatial resolution 
The spatial resolution of the PAM subsystem was measured experimentally by imaging a surgical blade. The lateral 
resolution of PAM, determined by the objective lens NA, is estimated to be ~3.9 µm. The axial resolution of PAM is 
measured to be ~17 µm, as limited by the 100 MHz bandwidth of the ultrasonic transducer. We quantified the spatial 
resolution of the FCM subsystem by imaging a mirror surface. The experimental data yields a lateral resolution of ~3.9 
µm and a axial resolution of ~38 µm (Table 1). 
 
Table 1.  Spatial resolution of the integrated PA-FCM system. 
 
Modality Transverse resolution Axial resolution Contrast 
Fluorescence confocal 
microscopy 
3.9 µm 17 µm Fluorescence 
Photoacoustic 
microscopy 
3.9 µm 38 µm Optical absorption 
 
 
2.3 In vivo mapping of sO2 and pO2 
Understanding of oxygen consumption and delivery in vivo requires the development of imaging techniques for mapping 
the intravascular oxygen concentration. Oxygen in blood is either dissolved or bound to hemoglobin. pO2 quantifies the 
concentration of dissolved oxygen which is directly available to cell; sO2 quantifies the amount of oxygen bound to 
hemoglobin. 
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PAM subsystem can measure sO2 by spectroscopically measuring hemoglobin absorption [1]. 
 ])[(])[()( 22 HbOHbr iHbOiHbria λελελμ +=  (1) 
where µa is the hemoglobin optical absorption measured by PAM, 2HbOε  and Hbrε  are the molar extinction coefficients 
of oxygenated and deoxygenated hemoglobin, and λ  is the laser wavelength. By a radiometric procedure  
 
][][
][
2
2
2 HbrHbO
HbOsO += , (2) 
sO2 value can be calculated pixel by pixel.  
FCM subsystem can measure pO2 using phosphorescence lifetime quenching technique [7-10]. Oxygen-sensitive Pd-
porphyrin phosphorescence probe is injected into the systemic vasculature. The phosphorescence is quenched by 
intravascular oxygen. The relationship of phosphorescence lifetime and the pO2 follows the Stern-Volmer equation 
 ][
11
2
0
pOkq+= ττ , (3) 
where 0τ is the phosphorescence lifetime without oxygen quenching and qk is the quenching constant calibrated in 
vitro[11]. In our studies, we used the oxygen-sensitive probe, Pd-meso-tetra (4-carboxyphenyl) porphyrin (PdT790, 
Frontier Scientific). It has a peak absorption wavelength of 524 nm. The PdT790 (10 mg/ml) was conjugated with 
bovine serum albumin (60 mg/ml) in 0.9% NaCl solution to prevent its leakage from the vasculature. We injected a 0.1 
ml volume of phosphorescent  probe solution through the tail vein. Images were acquired 10 min after injection to allow 
the probe to equilibrate in the blood. 
 
3. RESULTS AND DISCUSSION 
We imaged nude mouse ears to demonstrate the dual-modality microscopy of sO2 and pO2 in vivo. All experimental 
animal procedures were carried out in conformity with the laboratory animal protocol approved by the Animal Studies  
Committee of Washington University in St. Louis. 
The photoacoustic image of the mouse ear vasculature were acquired at two wavelengths of 570 nm and 578 nm. At the 
isosbestic wavelength of 570 nm (Fig. 2(a)), the 2HbO and Hbr  have the same molar extinction coefficients. Hence, 
the photoacoustic signal amplitudes measure the total hemoglobin concentration ( HbT ). Photoacoustic image at 578 
nm was also acquired. Aided by the molar extinction spectra of 2HbO and Hbr , intravascular sO2 level is computed as 
shown in Fig. 2(b). In the sO2 mapping, the arterioles and venules are pseudo-colored in red and blue based on their 
different sO2 levels [12].   
Figure 3 (a) shows the FCM image of the mouse ear after injection of the Pd-porphyrin probe. The sebaceous glands and 
vasculature can be seen in images as dot and tree features. The autofluorescence from the glands has nanosecond-scale 
lifetime while the phosphorescence lifetime of the Pd-porphyrin probe is ~100 µs. Thus we can separate the images  of 
sebaceous glands and vasculature by splitting the FCM signal in time domain. Figure 3(b) and 3(c) show the time-
integrated FCM images in the range of 0-5 µs and 5-500 µs. The latter one shows the blood vasculature based on the  
phosphorescence contrast. In the regions indicated by the arrows, the phosphorescence lifetime in artery (105 µs) is 
shorter than that in the vein (141 µs). Using Stern-Volmer equation, the shorter lifetime can be converted to a higher 
arterial pO2 level, which agrees with the known physiology. 
Fitting of the phosphorescence decay at each scanning step gives the lifetime mapping as shown in figure 4(a). 
Subsequently, mapping of intravascular pO2 (Fig. 4(b)) is generated using Eq. 3. In the pO2 mapping, the arterioles and 
venules can be distinguished by different pO2 levels.  
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Figure 2. PAM imaging of a mouse ear in vivo. (a) Photoacoustic image of total concentration of hemoglobin 
acquired at the isosbestic wavelength of 570 nm. (b) Photoacoustic image of sO2 mapping.  
 
 
Figure 3. FCM imaging of a mouse ear in vivo. (a) FCM image of time-integrated phosphorescence excited at 523 
nm. (b) FCM image of the sebaceous gland. (c) FCM image of the vasculature. FL: fluorescence.   
 
 
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Figure 4. FCM imaging of a mouse ear in vivo. (a) FCM image of phosphorescence lifetime mapping. (b) FCM 
image of pO2 mapping. 
  
In summary, we demonstrate that the multi-modal PA-FCM imaging system provides complementary photoacoustic and 
fluorescence contrasts. Intrinsically co-registered PAM and FCM images can be acquired. Intravascular sO2 and pO2 
levels, as well as total hemoglobin concentration, can be mapped vessel by vessel in vivo. Future study toward 
quantitative analysis of oxygen transport and consumption in tissues is very attractive. 
  
ACKNOWLEDGEMENTS   
We thank C. Zhang for helping with data processing and manuscript preparation, and Professor J. Ballard for careful 
proofreading. This work was sponsored in part by National Institutes of Health (NIH) grants R01 EB000712, R01 
EB008085, R01 CA134539, U54 CA136398, and 5P60 DK02057933. L. Wang has a financial interest in 
Microphotoacoustics, Inc., and Endra, Inc., which, however, did not support this work.  
 
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Integrated photoacoustic and fluorescence confocal microscopy

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Integrated photoacoustic and fluorescence confocal microscopy

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