The axial resolution of photoacoustic microscopy (PAM) is much lower than its lateral resolution, which resolves down to the submicron level. Here we achieved so far the highest axial resolution of 7.6 μm by using a commercial 125 MHz ultrasonic transducer for signal detection, followed by the Wiener deconvolution for signal processing. The axial resolution was validated by imaging two layers of red ink in a wedge shape. Melanoma cells were imaged ex vivo with high axial resolution. Compared with a PAM system with a 50 MHz ultrasonic transducer, our high-axial-resolution PAM system resolved the blood vessels in mouse ears in vivo much more clearly in the depth direction

Maslov, Konstantin

Wang, Lihong V.

Yao, Junjie

Zhang, Chi

Caltech Authors - Main

PROCEEDINGS OF SPIE
SPIEDigitalLibrary.org/conference-proceedings-of-spie
Photoacoustic microscopy with 7.6-µ
m axial resolution
Chi  Zhang, Konstantin  Maslov, Junjie  Yao, Lihong V.
Wang
Chi  Zhang, Konstantin  Maslov, Junjie  Yao, Lihong V. Wang, "Photoacoustic
microscopy with 7.6-µm axial resolution," Proc. SPIE 8581, Photons Plus
Ultrasound: Imaging and Sensing 2013, 85812W (4 March 2013); doi:
10.1117/12.2003248
Event: SPIE BiOS, 2013, San Francisco, California, United States
Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 9/5/2018  Terms of Use: https://www.spiedigitallibrary.org/terms-of-use
  
Photoacoustic microscopy with 7.6 µm axial resolution 
 
Chi Zhang, Konstantin Maslov, Junjie Yao, and Lihong V. Wang* 
Department of Biomedical Engineering, Washington University in St. Louis, One Brookings Drive, 
St. Louis, MO 63130, USA 
* Corresponding author: LHWANG@WUSTL.EDU 
ABSTRACT 
The axial resolution of photoacoustic microscopy (PAM) is much lower than its lateral resolution, which resolves down 
to the submicron level. Here we achieved so far the highest axial resolution of 7.6 µm by using a commercial 125 MHz 
ultrasonic transducer for signal detection, followed by the Wiener deconvolution for signal processing. The axial 
resolution was validated by imaging two layers of red ink in a wedge shape. Melanoma cells were imaged ex vivo with 
high axial resolution. Compared with a PAM system with a 50 MHz ultrasonic transducer, our high-axial-resolution 
PAM system resolved the blood vessels in mouse ears in vivo much more clearly in the depth direction. 
Keywords: photoacoustic microscopy, axial resolution, high-frequency ultrasonic transducer, deconvolution 
 
1. INTRODUCTION  
Photoacoustic microscopy has been demonstrated to image rich endogenous optical-absorption contrasts in vivo, 
including hemoglobin, melanin, DNA, RNA, cytochrome, myoglobin, bilirubin, water, and lipid.[1]–[8] At proper 
wavelengths, any molecules can potentially be imaged by PAM. Moreover, the functional parameters associated with 
these biomolecules can be imaged by PAM, such as hemoglobin oxygen saturation, blood flow speed, and temperature 
variation.[9]–[11] With these unique advantages, label-free PAM is expected to join the mainstream optical microscopy 
technologies. 
Spatial resolution is a key parameter of microscopy. For optical-resolution (OR-) PAM, the lateral resolution, provided 
by the optical focusing, has achieved the submicron level in both transmission and reflection modes.[12],[13] The axial 
resolution, provided by the time-resolved ultrasonic detection, is limited by the ultrasonic transducer bandwidth[14] 
(approximately proportional to the central frequency). Since higher-frequency ultrasound attenuates faster in biological 
tissues, increasing the bandwidth for better axial resolution will decrease the maximum imaging depth. So far, ~15 µm 
axial resolution for depths up to 1.2 mm has been reported,[15],[16] but the axial resolution still remains much lower than 
the lateral resolution in OR-PAM.  
Here we achieved so far the highest 7.6 µm axial resolution for PAM by using a commercial 125 MHz ultrasonic 
transducer for signal detection and the Wiener deconvolution method for signal processing. The high axial resolution 
was experimentally validated. As two example applications, we implemented high-axial-resolution imaging of 
melanoma cells ex vivo and mouse ears in vivo. 
2. MATERIALS AND METHODS 
Fig. 1 shows the high axial resolution PAM system. The laser (NT242-SH, Ekspla) generated laser pulses with a 
wavelength of 532 nm, a pulse width of 5 ns, and a repetition rate of 1 KHz. The laser pulses were focused onto the 
sample by a 0.32 NA objective, providing ~0.8 µm lateral resolution. The excited photoacoustic waves from the sample 
were detected by an ultrasonic transducer (125 MHz central frequency, 100 MHz bandwidth; V2062, Olympus NDT) 
with a focusing acoustic lens (0.8 NA). The photoacoustic signals were digitized at 1 GS/s and processed by a computer. 
The sample was mechanically scanned in 2D to generate a 3D image. 
For each A-line signal, the envelope is normally extracted to convert the photoacoustic signal to a depth-resolved image.  
However, as shown in the literature, the axial (depth) resolution can be further improved by deconvolution methods.[13] 
The photoacoustic signal can be approximately modeled as the convolution of the system impulse response and the 
depth-resolved target function. Deconvolving the photoacoustic signal with the system impulse response helps to recover 
Photons Plus Ultrasound: Imaging and Sensing 2013, edited by Alexander A. Oraevsky, Lihong V. Wang,
Proc. of SPIE Vol. 8581, 85812W · © 2013 SPIE · CCC code: 1605-7422/13/$18 · doi: 10.1117/12.2003248
Proc. of SPIE Vol. 8581  85812W-1
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the target function. In other words, deconvolution recovers the attenuated frequency components of the signal and 
broadens the system bandwidth, thereby improving the axial resolution (limited by the signal-to-noise ratio). Here the 
Wiener deconvolution method[17] was used to further enhance the axial resolution. 
 
 
Figure 1. System schematic of PAM. 
 
3. RESULTS AND DISCUSSION 
We designed an experiment to quantitatively measure the axial resolution of the PAM system. The sample to be imaged 
is shown in Fig. 2(a). The sample consists of two layers of red ink, one on the polymethylpentene (TPX) plastic (upper) 
and the other on the glass slide (lower). The two layers were placed in a wedge shape, providing continuously variable 
distance between the two layers. The top layer was smeared on the TPX plastic because the acoustic impedance of the 
TPX plastic is close to that of water. A B-scan image of the sample calculated is shown in Fig. 2(b). The image was 
calculated by the deconvolution method to further improve the axial resolution. The contrast-to-noise ratio (CNR) versus 
the distance between the two layers was calculated. The axial resolution is defined as the distance with 6 dB CNR. It can 
be calculated that the axial resolution of the PAM system is 7.6 μm. Fig. 2(c) shows the profile across the B-scan image 
where the distance between the two layers is 7.6 μm. 
We used the high-axial-resolution PAM system to image melanoma cells ex vivo. The cells were seeded onto a slide at a 
density of 30 mm-2 and fixed by formalin. The PAM image of a melanoma cell is rendered in 3D as a video (Video 3). 
The bright dots in the PAM images are melanosomes, the organelles containing melanin. The 3D structure of the cell 
was resolved in both lateral and axial directions. Note that here PAM generates a 3D image without depth scanning, 
which, however, is required in confocal microscopy and two photon microscopy. 
 
Ultrasonic 
transducer
Sample
Objective
Preamplifier
Computer
Laser
Water
Condenser 
lens
Mirror
Pinhole
Proc. of SPIE Vol. 8581  85812W-2
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.-; ..;
or)tical absorption
 
 
Figure 2. (a) 
sample. (c) T
 
Video 3. 3D 
 
The sample to
he profile acro
PAM image o
 be imaged co
ss the B-scan 
f a melanoma c
0
0.2
0.4
0.6
0.8
1
N
or
m
al
iz
ed
 P
A 
am
pl
itu
de
Re
(a)
 
(b)
(c)
nsists of two 
image where t
ell ex vivo. htt
0 10
D
d ink
layers of red i
he distance be
p://dx.doi.org/
20
epth (μm)
TPX plas
7.6 µm
6 dB C
7.6 µm
nk in a wedge
tween the two 
 
doi.number.go
30 40
Glass s
tic
 
O
pt
ic
al
ab
so
rp
tio
n
0
1
 with 
NR
10 µm
 
 shape. (b) Th
layers is 7.6 μ
es.here 
lide
O
pt
ic
al
 a
bs
or
pt
io
n
e B-scan imag
m. 
e of the 
Proc. of SPIE Vol. 8581  85812W-3
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PPI
mu
ali
0
II
r
(d)
i
i
1
o
a
ó
i
 
 
By imaging 
MHz ultraso
transmission 
expected to b
systems. Fig
clearly by the
 
Figure 4. B-
transducers. 
 
We analyzed
ultrasonic tra
surface laser
safety limit o
to further im
may be quit
frequency.[19
 
mouse ears in
nic transduce
mode to prov
e >2 times. F
s. 4(c) and (4d
 high-axial-re
scan PAM im
 the detection
nsducer. In th
 fluence was 
f 20 mJ/cm2. S
prove the axia
e limited beca
] 
 vivo, we com
r (90% band
ide a fair co
igs. 4(a) and 4
) show anothe
solution PAM 
ages of a mo
 sensitivity o
e in vivo mou
estimated to b
o the 125 MH
l resolution by
use in most 
pared the high
width). The 
mparison. Th
(b) show a pa
r pair of imag
system with th
use ear in viv
f our PAM sy
se ear imagin
e 6.5 mJ/cm2,
z ultrasonic tr
 simply using
soft tissues th
-axial-resolut
system with 
e difference b
ir of B-scan im
es. It can be s
e 125 MHz ul
o acquired w
stem, which
g experiment, 
 well below t
ansducer is su
 a higher-freq
e attenuation 
ion PAM with
the lower-fre
etween the tw
ages of an ea
een that the bl
trasonic transd
ith (a,c) 50 M
is a major co
the laser puls
he American 
itable for in vi
uency ultrason
coefficient is 
 a PAM syste
quency transd
o systems in
r at the same 
ood vessels ar
ucer. 
Hz and (b,d
ncern when u
e energy was 
National Stand
vo blood vess
ic transducer
nearly propor
m[18] which u
ucer also wo
 axial resolut
position from
e resolved mu
 
) 125 MHz u
sing a high-fr
about 150 nJ,
ards Institute
el imaging. If 
, the penetratio
tional to the 
ses a 50 
rked in 
ion was 
 the two 
ch more 
ltrasonic 
equency 
 and the 
 (ANSI) 
we want 
n depth 
acoustic 
Proc. of SPIE Vol. 8581  85812W-4
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4. CONCLUSIONS 
An axial resolution of 7.6 µm, the finest thus far, has been achieved for PAM. The results were validated by phantom 
experiments, ex vivo cell imaging and in vivo mouse ear imaging. The improved axial resolution benefits PAM in high-
resolution 3D imaging. 
The improvement of axial resolution was largely contributed by the deconvolution method. With higher SNR achieved 
by technology development, we expect to achieve an even better axial resolution. However, note that deconvolution 
should be applied to a linear and shift-invariant system. In PAM, the linearity holds well with low laser intensity while 
absorption saturation or nonlinear thermal expansion can be ignored.[20] The shift invariance holds accurately within the 
focal zone of the ultrasonic transducer. 
ACKNOWLEDGMENT 
This work was sponsored in part by National Institutes of Health grants R01 EB000712, R01 EB008085, R01 
CA134539, U54 CA136398, R01 CA157277, and R01 CA159959. L.W. has a financial interest in Microphotoacoustics, 
Inc. and Endra, Inc., which, however, did not support this work. K.M. has a financial interest in Microphotoacoustics, 
Inc. 
REFERENCES 
[1] H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Functional photoacoustic microscopy for high-resolution and 
noninvasive in vivo imaging,” Nat. Biotech. 24(7), 848–851 (2006). 
[2] D.-K. Yao, K. Maslov, K. K. Shung, Q. Zhou, and L. V. Wang, “In vivo label-free photoacoustic microscopy of cell 
nuclei by excitation of DNA and RNA,” Opt. Lett. 35(24), 4139–4141 (2010). 
[3] C. Zhang, Y. S. Zhang, D.-K. Yao, Y. Xia, and L. V. Wang, “Label-free photoacoustic microscopy of cytochromes,” 
J. Biomed. Opt. 18(2), 020504 (2013). 
[4] C. Zhang, Y.-J. Cheng, J. Chen, S. Wickline, and L. V. Wang, “Label-free photoacoustic microscopy of myocardial 
sheet architecture,” J. Biomed. Opt. 17(6), 060506 (2012). 
[5] Y. Zhou, C. Zhang, D.-K. Yao, and L. V. Wang, “Photoacoustic microscopy of bilirubin,” J. Biomed. Opt. 17(12), 
126019 (2012). 
[6] Z. Xu, Q. Zhu, and L. V. Wang, “In vivo photoacoustic tomography of mouse cerebral edema induced by cold 
injury,” J. Biomed. Opt. 16(6), 066020 (2011). 
[7] H.-W. Wang, N. Chai, P. Wang, S. Hu, W. Dou, et al., “Label-free bond-selective imaging by listening to 
vibrationally excited molecules,” Phy. Rev. Lett. 106(23), 238106 (2011). 
[8] T. J. Allen, A. Hall, A. P. Dhillon, J. S. Owen, and P. C. Beard, “Spectroscopic photoacoustic imaging of lipid-rich 
plaques in the human aorta in the 740 to 1400 nm wavelength range,” J. Biomed. Opt. 17(6), 061209 (2012). 
[9] J. Yao, K. Maslov, Y. Zhang, Y. Xia, and L. V. Wang, “Label-free oxygen-metabolic photoacoustic microscopy in 
vivo,” J. Biomed. Opt. 16(7), 076003 (2011). 
[10] Y. Wang, K. Maslov, and L. V. Wang, “Spectrally encoded photoacoustic microscopy using a digital mirror device,” 
J. Biomed. Opt. 17(6), 066020 (2012). 
[11] J. Shah, S. Park, S. Aglyamov, T. Larson, L. Ma, et al., “Photoacoustic imaging and temperature measurement for 
photothermal cancer therapy,” J. Biomed. Opt. 13(3), 034024 (2008). 
[12] C. Zhang, K. Maslov, and L. V. Wang, “Subwavelength-resolution label-free photoacoustic microscopy of optical 
absorption in vivo,” Opt. Lett. 35(19), 3195–3197 (2010). 
[13] C. Zhang, K. Maslov, S. Hu, R. Chen, Q. Zhou, et al., “Reflection-mode submicron-resolution in vivo photoacoustic 
microscopy,” J. Biomed. Opt. 17(2), 020501 (2012). 
[14] C. Zhang, K. Maslov, J. Yao, and L. V. Wang, “In vivo photoacoustic microscopy with 7.6-µm axial resolution 
using a commercial 125-MHz ultrasonic transducer,” J. Biomed. Opt. 17(11), 116016 (2012). 
[15] L. Wang, K. Maslov, J. Yao, B. Rao, and L. V. Wang, “Fast voice-coil scanning optical-resolution photoacoustic 
microscopy,” Opt. Lett. 36(2), 139–141 (2011). 
[16] Y. Wang, K. Maslov, C. Kim, S. Hu, and L. V. Wang, “Integrated photoacoustic and fluorescence confocal 
microscopy,” IEEE Trans. Biomed. Eng. 57(10), 2576–2578 (2010). 
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[17] J. A. Jensen, J. Mathorne, T. Gravesen, and B. Stage, “Deconvolution of in-vivo ultrasound B-mode images,” 
Ultrason. Imag. 15(2), 122–133 (1993). 
[18] J. Yao, K. Maslov, E. R. Puckett, K. J. Rowland, B. W. Warner, et al., “Double-illumination photoacoustic 
microscopy,” Opt. Lett. 37(4), 659–662 (2012). 
[19] P. T. Wells, Biomedical Ultrasonics, Academic Press, New York, pp. 120–123 (1977). 
[20] A. Danielli, C. P. Favazza, K. Maslov, and L. V. Wang, “Picosecond absorption relaxation measured with 
nanosecond laser photoacoustics,” Appl. Phys. Lett. 97(16), 163701 (2010). 
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Photoacoustic microscopy with 7.6-μm axial resolution

PROCEEDINGS OF SPIE
SPIEDigitalLibrary.org/conference-proceedings-of-spie
Photoacoustic microscopy with 7.6-µ
m axial resolution
Chi  Zhang, Konstantin  Maslov, Junjie  Yao, Lihong V.
Wang
Chi  Zhang, Konstantin  Maslov, Junjie  Yao, Lihong V. Wang, "Photoacoustic
microscopy with 7.6-µm axial resolution," Proc. SPIE 8581, Photons Plus
Ultrasound: Imaging and Sensing 2013, 85812W (4 March 2013); doi:
10.1117/12.2003248
Event: SPIE BiOS, 2013, San Francisco, California, United States
Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 9/5/2018  Terms of Use: https://www.spiedigitallibrary.org/terms-of-use
 
 
Photoacoustic microscopy with 7.6 µm axial resolution 
 
Chi Zhang, Konstantin Maslov, Junjie Yao, and Lihong V. Wang* 
Department of Biomedical Engineering, Washington University in St. Louis, One Brookings Drive, 
St. Louis, MO 63130, USA 
* Corresponding author: LHWANG@WUSTL.EDU 
ABSTRACT 
The axial resolution of photoacoustic microscopy (PAM) is much lower than its lateral resolution, which resolves down 
to the submicron level. Here we achieved so far the highest axial resolution of 7.6 µm by using a commercial 125 MHz 
ultrasonic transducer for signal detection, followed by the Wiener deconvolution for signal processing. The axial 
resolution was validated by imaging two layers of red ink in a wedge shape. Melanoma cells were imaged ex vivo with 
high axial resolution. Compared with a PAM system with a 50 MHz ultrasonic transducer, our high-axial-resolution 
PAM system resolved the blood vessels in mouse ears in vivo much more clearly in the depth direction. 
Keywords: photoacoustic microscopy, axial resolution, high-frequency ultrasonic transducer, deconvolution 
 
1. INTRODUCTION  
Photoacoustic microscopy has been demonstrated to image rich endogenous optical-absorption contrasts in vivo, 
including hemoglobin, melanin, DNA, RNA, cytochrome, myoglobin, bilirubin, water, and lipid.[1]–[8] At proper 
wavelengths, any molecules can potentially be imaged by PAM. Moreover, the functional parameters associated with 
these biomolecules can be imaged by PAM, such as hemoglobin oxygen saturation, blood flow speed, and temperature 
variation.[9]–[11] With these unique advantages, label-free PAM is expected to join the mainstream optical microscopy 
technologies. 
Spatial resolution is a key parameter of microscopy. For optical-resolution (OR-) PAM, the lateral resolution, provided 
by the optical focusing, has achieved the submicron level in both transmission and reflection modes.[12],[13] The axial 
resolution, provided by the time-resolved ultrasonic detection, is limited by the ultrasonic transducer bandwidth[14] 
(approximately proportional to the central frequency). Since higher-frequency ultrasound attenuates faster in biological 
tissues, increasing the bandwidth for better axial resolution will decrease the maximum imaging depth. So far, ~15 µm 
axial resolution for depths up to 1.2 mm has been reported,[15],[16] but the axial resolution still remains much lower than 
the lateral resolution in OR-PAM.  
Here we achieved so far the highest 7.6 µm axial resolution for PAM by using a commercial 125 MHz ultrasonic 
transducer for signal detection and the Wiener deconvolution method for signal processing. The high axial resolution 
was experimentally validated. As two example applications, we implemented high-axial-resolution imaging of 
melanoma cells ex vivo and mouse ears in vivo. 
2. MATERIALS AND METHODS 
Fig. 1 shows the high axial resolution PAM system. The laser (NT242-SH, Ekspla) generated laser pulses with a 
wavelength of 532 nm, a pulse width of 5 ns, and a repetition rate of 1 KHz. The laser pulses were focused onto the 
sample by a 0.32 NA objective, providing ~0.8 µm lateral resolution. The excited photoacoustic waves from the sample 
were detected by an ultrasonic transducer (125 MHz central frequency, 100 MHz bandwidth; V2062, Olympus NDT) 
with a focusing acoustic lens (0.8 NA). The photoacoustic signals were digitized at 1 GS/s and processed by a computer. 
The sample was mechanically scanned in 2D to generate a 3D image. 
For each A-line signal, the envelope is normally extracted to convert the photoacoustic signal to a depth-resolved image.  
However, as shown in the literature, the axial (depth) resolution can be further improved by deconvolution methods.[13] 
The photoacoustic signal can be approximately modeled as the convolution of the system impulse response and the 
depth-resolved target function. Deconvolving the photoacoustic signal with the system impulse response helps to recover 
Photons Plus Ultrasound: Imaging and Sensing 2013, edited by Alexander A. Oraevsky, Lihong V. Wang,
Proc. of SPIE Vol. 8581, 85812W · © 2013 SPIE · CCC code: 1605-7422/13/$18 · doi: 10.1117/12.2003248
Proc. of SPIE Vol. 8581  85812W-1
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Terms of Use: https://www.spiedigitallibrary.org/terms-of-use
 
 
the target function. In other words, deconvolution recovers the attenuated frequency components of the signal and 
broadens the system bandwidth, thereby improving the axial resolution (limited by the signal-to-noise ratio). Here the 
Wiener deconvolution method[17] was used to further enhance the axial resolution. 
 
 
Figure 1. System schematic of PAM. 
 
3. RESULTS AND DISCUSSION 
We designed an experiment to quantitatively measure the axial resolution of the PAM system. The sample to be imaged 
is shown in Fig. 2(a). The sample consists of two layers of red ink, one on the polymethylpentene (TPX) plastic (upper) 
and the other on the glass slide (lower). The two layers were placed in a wedge shape, providing continuously variable 
distance between the two layers. The top layer was smeared on the TPX plastic because the acoustic impedance of the 
TPX plastic is close to that of water. A B-scan image of the sample calculated is shown in Fig. 2(b). The image was 
calculated by the deconvolution method to further improve the axial resolution. The contrast-to-noise ratio (CNR) versus 
the distance between the two layers was calculated. The axial resolution is defined as the distance with 6 dB CNR. It can 
be calculated that the axial resolution of the PAM system is 7.6 μm. Fig. 2(c) shows the profile across the B-scan image 
where the distance between the two layers is 7.6 μm. 
We used the high-axial-resolution PAM system to image melanoma cells ex vivo. The cells were seeded onto a slide at a 
density of 30 mm-2 and fixed by formalin. The PAM image of a melanoma cell is rendered in 3D as a video (Video 3). 
The bright dots in the PAM images are melanosomes, the organelles containing melanin. The 3D structure of the cell 
was resolved in both lateral and axial directions. Note that here PAM generates a 3D image without depth scanning, 
which, however, is required in confocal microscopy and two photon microscopy. 
 
Ultrasonic 
transducer
Sample
Objective
Preamplifier
Computer
Laser
Water
Condenser 
lens
Mirror
Pinhole
Proc. of SPIE Vol. 8581  85812W-2
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. -; ..;
or)tical absorption
 
 
Figure 2. (a) 
sample. (c) T
 
Video 3. 3D 
 
The sample to
he profile acro
PAM image o
 be imaged co
ss the B-scan 
f a melanoma c
0
0.2
0.4
0.6
0.8
1
N
or
m
al
iz
ed
 P
A 
am
pl
itu
de
Re
(a)
 
(b)
(c)
nsists of two 
image where t
ell ex vivo. htt
0 10
D
d ink
layers of red i
he distance be
p://dx.doi.org/
20
epth (μm)
TPX plas
7.6 µm
6 dB C
7.6 µm
nk in a wedge
tween the two 
 
doi.number.go
30 40
Glass s
tic
 
O
pt
ic
al
ab
so
rp
tio
n
0
1
 with 
NR
10 µm
 
 shape. (b) Th
layers is 7.6 μ
es.here 
lide
O
pt
ic
al
 a
bs
or
pt
io
n
e B-scan imag
m. 
e of the 
Proc. of SPIE Vol. 8581  85812W-3
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PPI
mu
ali
0
II
r
(d)
i
i
1
o
a
ó
i
 
 
By imaging 
MHz ultraso
transmission 
expected to b
systems. Fig
clearly by the
 
Figure 4. B-
transducers. 
 
We analyzed
ultrasonic tra
surface laser
safety limit o
to further im
may be quit
frequency.[19
 
mouse ears in
nic transduce
mode to prov
e >2 times. F
s. 4(c) and (4d
 high-axial-re
scan PAM im
 the detection
nsducer. In th
 fluence was 
f 20 mJ/cm2. S
prove the axia
e limited beca
] 
 vivo, we com
r (90% band
ide a fair co
igs. 4(a) and 4
) show anothe
solution PAM 
ages of a mo
 sensitivity o
e in vivo mou
estimated to b
o the 125 MH
l resolution by
use in most 
pared the high
width). The 
mparison. Th
(b) show a pa
r pair of imag
system with th
use ear in viv
f our PAM sy
se ear imagin
e 6.5 mJ/cm2,
z ultrasonic tr
 simply using
soft tissues th
-axial-resolut
system with 
e difference b
ir of B-scan im
es. It can be s
e 125 MHz ul
o acquired w
stem, which
g experiment, 
 well below t
ansducer is su
 a higher-freq
e attenuation 
ion PAM with
the lower-fre
etween the tw
ages of an ea
een that the bl
trasonic transd
ith (a,c) 50 M
is a major co
the laser puls
he American 
itable for in vi
uency ultrason
coefficient is 
 a PAM syste
quency transd
o systems in
r at the same 
ood vessels ar
ucer. 
Hz and (b,d
ncern when u
e energy was 
National Stand
vo blood vess
ic transducer
nearly propor
m[18] which u
ucer also wo
 axial resolut
position from
e resolved mu
 
) 125 MHz u
sing a high-fr
about 150 nJ,
ards Institute
el imaging. If 
, the penetratio
tional to the 
ses a 50 
rked in 
ion was 
 the two 
ch more 
ltrasonic 
equency 
 and the 
 (ANSI) 
we want 
n depth 
acoustic 
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4. CONCLUSIONS 
An axial resolution of 7.6 µm, the finest thus far, has been achieved for PAM. The results were validated by phantom 
experiments, ex vivo cell imaging and in vivo mouse ear imaging. The improved axial resolution benefits PAM in high-
resolution 3D imaging. 
The improvement of axial resolution was largely contributed by the deconvolution method. With higher SNR achieved 
by technology development, we expect to achieve an even better axial resolution. However, note that deconvolution 
should be applied to a linear and shift-invariant system. In PAM, the linearity holds well with low laser intensity while 
absorption saturation or nonlinear thermal expansion can be ignored.[20] The shift invariance holds accurately within the 
focal zone of the ultrasonic transducer. 
ACKNOWLEDGMENT 
This work was sponsored in part by National Institutes of Health grants R01 EB000712, R01 EB008085, R01 
CA134539, U54 CA136398, R01 CA157277, and R01 CA159959. L.W. has a financial interest in Microphotoacoustics, 
Inc. and Endra, Inc., which, however, did not support this work. K.M. has a financial interest in Microphotoacoustics, 
Inc. 
REFERENCES 
[1] H. F. Zhang, K. Maslov, G. Stoica, and L. V. Wang, “Functional photoacoustic microscopy for high-resolution and 
noninvasive in vivo imaging,” Nat. Biotech. 24(7), 848–851 (2006). 
[2] D.-K. Yao, K. Maslov, K. K. Shung, Q. Zhou, and L. V. Wang, “In vivo label-free photoacoustic microscopy of cell 
nuclei by excitation of DNA and RNA,” Opt. Lett. 35(24), 4139–4141 (2010). 
[3] C. Zhang, Y. S. Zhang, D.-K. Yao, Y. Xia, and L. V. Wang, “Label-free photoacoustic microscopy of cytochromes,” 
J. Biomed. Opt. 18(2), 020504 (2013). 
[4] C. Zhang, Y.-J. Cheng, J. Chen, S. Wickline, and L. V. Wang, “Label-free photoacoustic microscopy of myocardial 
sheet architecture,” J. Biomed. Opt. 17(6), 060506 (2012). 
[5] Y. Zhou, C. Zhang, D.-K. Yao, and L. V. Wang, “Photoacoustic microscopy of bilirubin,” J. Biomed. Opt. 17(12), 
126019 (2012). 
[6] Z. Xu, Q. Zhu, and L. V. Wang, “In vivo photoacoustic tomography of mouse cerebral edema induced by cold 
injury,” J. Biomed. Opt. 16(6), 066020 (2011). 
[7] H.-W. Wang, N. Chai, P. Wang, S. Hu, W. Dou, et al., “Label-free bond-selective imaging by listening to 
vibrationally excited molecules,” Phy. Rev. Lett. 106(23), 238106 (2011). 
[8] T. J. Allen, A. Hall, A. P. Dhillon, J. S. Owen, and P. C. Beard, “Spectroscopic photoacoustic imaging of lipid-rich 
plaques in the human aorta in the 740 to 1400 nm wavelength range,” J. Biomed. Opt. 17(6), 061209 (2012). 
[9] J. Yao, K. Maslov, Y. Zhang, Y. Xia, and L. V. Wang, “Label-free oxygen-metabolic photoacoustic microscopy in 
vivo,” J. Biomed. Opt. 16(7), 076003 (2011). 
[10] Y. Wang, K. Maslov, and L. V. Wang, “Spectrally encoded photoacoustic microscopy using a digital mirror device,” 
J. Biomed. Opt. 17(6), 066020 (2012). 
[11] J. Shah, S. Park, S. Aglyamov, T. Larson, L. Ma, et al., “Photoacoustic imaging and temperature measurement for 
photothermal cancer therapy,” J. Biomed. Opt. 13(3), 034024 (2008). 
[12] C. Zhang, K. Maslov, and L. V. Wang, “Subwavelength-resolution label-free photoacoustic microscopy of optical 
absorption in vivo,” Opt. Lett. 35(19), 3195–3197 (2010). 
[13] C. Zhang, K. Maslov, S. Hu, R. Chen, Q. Zhou, et al., “Reflection-mode submicron-resolution in vivo photoacoustic 
microscopy,” J. Biomed. Opt. 17(2), 020501 (2012). 
[14] C. Zhang, K. Maslov, J. Yao, and L. V. Wang, “In vivo photoacoustic microscopy with 7.6-µm axial resolution 
using a commercial 125-MHz ultrasonic transducer,” J. Biomed. Opt. 17(11), 116016 (2012). 
[15] L. Wang, K. Maslov, J. Yao, B. Rao, and L. V. Wang, “Fast voice-coil scanning optical-resolution photoacoustic 
microscopy,” Opt. Lett. 36(2), 139–141 (2011). 
[16] Y. Wang, K. Maslov, C. Kim, S. Hu, and L. V. Wang, “Integrated photoacoustic and fluorescence confocal 
microscopy,” IEEE Trans. Biomed. Eng. 57(10), 2576–2578 (2010). 
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[17] J. A. Jensen, J. Mathorne, T. Gravesen, and B. Stage, “Deconvolution of in-vivo ultrasound B-mode images,” 
Ultrason. Imag. 15(2), 122–133 (1993). 
[18] J. Yao, K. Maslov, E. R. Puckett, K. J. Rowland, B. W. Warner, et al., “Double-illumination photoacoustic 
microscopy,” Opt. Lett. 37(4), 659–662 (2012). 
[19] P. T. Wells, Biomedical Ultrasonics, Academic Press, New York, pp. 120–123 (1977). 
[20] A. Danielli, C. P. Favazza, K. Maslov, and L. V. Wang, “Picosecond absorption relaxation measured with 
nanosecond laser photoacoustics,” Appl. Phys. Lett. 97(16), 163701 (2010). 
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Photoacoustic microscopy with 7.6-μm axial resolution

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