Handheld optical-resolution

photoacoustic microscopy

Li Lin
Pengfei Zhang
Song Xu
Junhui Shi
Lei Li
Junjie Yao
Lidai Wang
Jun Zou
Lihong V. Wang

Li Lin, Pengfei Zhang, Song Xu, Junhui Shi, Lei Li, Junjie Yao, Lidai Wang, Jun Zou, Lihong V. Wang,
“Handheld optical-resolution photoacoustic microscopy,” J. Biomed. Opt. 22(4), 041002 (2016),
doi: 10.1117/1.JBO.22.4.041002.
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 Journal of Biomedical Optics 22(4), 041002 (April 2017)

Handheld optical-resolution photoacoustic

microscopy
Li Lin,a,† Pengfei Zhang,a,† Song Xu,b Junhui Shi,a Lei Li,a Junjie Yao,a Lidai Wang,a,‡ Jun Zou,b and
Lihong V. Wanga,*
a
Washington University in St. Louis, Optical Imaging Laboratory, Department of Biomedical Engineering, One Brookings Drive, St. Louis,
Missouri 63130, United States
b
Texas A&M University, Institute for Solid State Electronics, Electrical Engineering Department, 400 Bizzell Street, College Station,
Texas 77840, United States

Abstract. Optical-resolution photoacoustic microscopy (OR-PAM) offers label-free in vivo imaging with high
spatial resolution by acoustically detecting optical absorption contrasts via the photoacoustic effect. We devel-
oped a compact handheld OR-PAM probe for fast photoacoustic imaging. Different from benchtop microscopes,
the handheld probe provides flexibility in imaging various anatomical sites. Resembling a cup in size, the probe
uses a two-axis water-immersible microelectromechanical system mirror to scan both the illuminating optical
beam and resultant acoustic beam. The system performance was tested in vivo by imaging the capillary
bed in a mouse ear and both the capillary bed and a mole on a human volunteer. © 2016 Society of Photo-Optical
Instrumentation Engineers (SPIE) [DOI: 10.1117/1.JBO.22.4.041002]

Keywords: photoacoustic handheld probe; optical-resolution photoacoustic microscopy; two-axis microelectromechanical system mir-
ror; confocal scanning; human skin imaging.
Paper 160264SSR received Apr. 21, 2016; accepted for publication Jun. 9, 2016; published online Oct. 24, 2016.

foci with a one-axis water-immersible microelectromechanical

The American Cancer Society recommends regular examina- system (MEMS) and by slow scanning the samples along the
tions of skin lesions as the best way to find skin cancers orthogonal directions.19
early.1 Therefore, a noninvasive device that can easily scan In this article, we present a handheld probe, based on
the body would facilitate such routine examinations. Optical OR-PAM, that uses a newly developed two-axis water-immers-
microscopy has excellent imaging contrast in soft tissue2 and ible MEMS scanning mirror.20 In the OR-PAM probe, the opti-
has been miniaturized for clinical applications. For use in medi- cal and acoustic beams are confocally configured to maximize
cal offices and clinics, handheld microscopes have been devel- the signal-to-noise ratio (SNR).10,21,22 The two beams are fast
oped based on various optical imaging modalities.3–6 Compact scanned by the MEMS mirror, yielding a 3-D imaging rate
confocal microscopy achieves fast imaging speed with high spa- of 2 Hz over a 2.5 × 2.0 × 0.5 mm3 volume. This probe was
tial resolution, but its shallow imaging depth (200 to 300 μm) tested in vivo on both the mouse ear and human skin.
limits its wide application.3,4 Optical coherence tomography- Figure 1(a) is a schematic of the optical and acoustic ele-
based probes exploit the optical contrast in backscattered ments in the OR-PAM handheld probe. A laser beam is directed
light5,6 and are complementary to photoacoustic tomography into the probe through a single-mode fiber (P1-460B-FC-2,
(PAT), which shows the optical absorption contrast. Thorlabs), which guides the light to a lens tube in the handheld
PAT is a hybrid imaging modality in which laser-generated probe. Two optical lenses (AC127-025-A, Thorlabs; and
ultrasound waves are used to obtain three-dimensional (3-D) PAC025, Newport) in the lens tube focus the light to an opti-
images of soft tissue.7,8 A major implementation of PAT, opti- cal–acoustic beam combiner immersed in water. To seal the
cal-resolution photoacoustic microscopy (OR-PAM), provides lens tube from water, we sealed the second lens at the end of
optical absorption contrast with high resolution9 and has been the lens tube with silicone caulk. The beam combiner, composed
proven capable of anatomical, chemical, functional, and meta- of an aluminum-coated prism (MRA10-F01, Thorlabs) and an
bolic imaging.10–15 By tightly focusing the laser beam to a depth uncoated prism (PS910, Thorlabs), provides acoustic and opti-
within the optical diffusion limit, OR-PAM provides capillary- cal coaxial alignments. The thin aluminum coating reflects light,
level spatial resolution.10 Recently, an OR-PAM system used a but transmits sound.22 The focused laser beam is reflected by the
water-immersion optical focusing lens with a numerical aperture MEMS mirror plate onto the surface of the object to be imaged.
of 1.23 to improve the spatial resolution to 220 nm.16 The im- The resultant photoacoustic waves are reflected by the MEMS
aging depth or thickness has also been improved by using near- mirror plate to an acoustic lens (LC1975, Thorlabs) that is
infrared light17 or double illumination.18 High-speed OR-PAM attached to the right side of the combiner. The photoacoustic
has been achieved by fast scanning the optical and acoustic waves are then detected by a 50-MHz ultrasound transducer
(V214-BB-RM, Olympus-NDT), which is tightly attached to
the beam combiner from the left. Thus, the laser beam and
*Address all correspondence to: Lihong V. Wang, E-mail: lhwang@biomed.
wustl.edu acoustic beam are confocally aligned through the beam com-
†
biner and the MEMS scanning mirror. An optical correction
These authors contributed equally to this work.
‡
Present address: City University of Hong Kong, Department of Mechanical and
Biomedical Engineering, Tat Chee Avenue, Kowloon, Hong Kong SAR 1083-3668/2016/$25.00 © 2016 SPIE

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 Lin et al.: Handheld optical-resolution photoacoustic microscopy

lens (45-119, Edmund) is attached to the top surface of the com- To characterize the lateral resolution of the handheld
biner to correct prism-induced aberration.10 Volumetric imaging OR-PAM system, the edge of a sharp blade was imaged in
is provided by fast rotational scanning along the x-axis and slow water, with a step size of 1 μm and a scanning range of
rotational scanning along the y-axis, as well as by the time- 250 μm along the x-axis. After conversion from polar coordi-
resolved detection of the ultrasound signal. The imaging nates to Cartesian coordinates, the maximum amplitude projec-
probe is filled with water for ultrasound coupling. tion (MAP) of the B-scan along the acoustic axis was calculated
Figure 1(b) is a 3-D rendering of the OR-PAM handheld and is plotted as a function of the displacement of the optical and
probe. The probe has dimensions of 80 mm × 115 mm × acoustic foci from the edge (Fig. 2). Assuming a Gaussian line
150 mm along the x, y, and z axes. The lens tube is mounted spread function, fitting of the measured data to an error function
on a five-axis lens positioner (LP-05A, Newport), which is indicates a lateral resolution of 5.0 μm (FWHM), which agrees
used for fine tuning the laser beam. The MEMS scanning mir- well with the theoretical diffraction-limited focused laser spot
ror is fixed on a compact linear stage (DS25-XY, Newport) for size (4.9 μm). The lateral resolution in tissue decreases with im-
precise position adjustment. The front, bottom, and cover pan- aging depth due to optical scattering.17 The axial resolution of
els are made of transparent acrylic to help to localize the tar- the system is determined by the bandwidth of the ultrasound
geted area. The imaging window on the bottom panel has a transducer and was estimated to be 26 μm. The penetration
diameter of 6 mm and is sealed with a transparent film. A depth of the handheld probe was quantified by a hair embedded
clamp mounted on the top of the probe protects the fiber in an optical scattering medium consisting of 2% Intralipid sol-
from excessive bending. The PA signal from the ultrasound ution (reduced scattering coefficient μs0 ¼ 15 cm−1 at 532 nm)
transducer is amplified by two integrated amplifiers (ZX60- and 3% agar gel, which mimics the reduced scattering coeffi-
43-S+ and ZFL-500LN+, Minicircuits) that provide a total cient of the human skin.23 The x-z projected MAP image is
amplification of 41 dB. The probe is grounded through a shown in Fig. 2(b). The PA signal from the hair 0.54 mm
cable attached to the backboard. beneath the surface (indicated by the dashed line) was still
The handheld probe is connected to the main system includ- detectable when 100-nJ pulse energy was used.
ing the laser, MEMS driver, data acquisition (DAQ) devices, and To demonstrate the imaging capability of the handheld
computer. The light is provided by a fiber laser (VPFL-G-10, OR-PAM probe, blood vessels in a nude mouse ear were imaged
VGEN) that generates 5-ns pulses at 532 nm, with a pulse rep- in vivo. All experimental animal procedures followed the labo-
etition rate of 88 kHz. The MEMS mirror is driven by a sinus- ratory animal protocol approved by the Animal Studies
oidal current from a homemade MEMS driver. The amplified Committee of Washington University in St. Louis. The mouse
PA signal from the probe is recorded by the DAQ device ear was acoustically coupled beneath the imaging window by
(ATS9350, Alazar Technologies) with a sampling rate of ultrasound gel. The round-trip scanning frequency of the MEMS
250 MHz. A low-pass filter (BLP-70+, Minicircuits) is con- mirror was set at 220 Hz for the fast axis and 1 Hz for the slow
nected between the amplifier and the analog-to-digital converter axis. Then, a region of 2.5 mm × 2.0 mm was imaged with a
to eliminate high-frequency noises. Both the mirror scanning volumetric imaging rate of 2 Hz (bidirectional scanning). The
and the DAQ are synchronized with the laser pulse. laser pulse energy was measured as 130 nJ under the imaging

Fig. 1 Schematic of the OR-PAM handheld probe. (a) 2-D sketch of the optical and acoustic beams in
the probe. UT, ultrasound transducer; AC, aluminum coating; AL, acoustic lens. (b) 3-D rendering of the
OR-PAM handheld probe. The front and the left side panels are removed for better visualization.

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 Lin et al.: Handheld optical-resolution photoacoustic microscopy

Fig. 2 Characteristics of the handheld OR-PAM probe. (a) Lateral

resolution measurement by imaging a sharp edge. Solid squares:
the PA amplitude when the laser beam was scanned across the
edge. Solid curve: the fit of the measured data to an error function.
Dashed line: the extracted Gaussian-shaped line spread function.
(b) Penetration depth measured by a hair embedded in an optical Fig. 4 Hemoglobin concentration monitoring in the vessels in a mouse
scattering medium mimicking skin tissue. The acoustic attenuation ear after tail vein injection of 0.9% saline. (a) Representative images
has been compensated for by multiplying the PA signal by a depth- recorded at different time points after the injection of 0.6 mL saline.
dependent exponential correction factor. The gray bar shows the nor- The stamps on the images indicate the time (min:s) after the injection.
malized PA signal amplitude. (b) The signal from the blood vessels within the dashed box in (a) as a
function of time. The injection procedure lasted 75 s.

process. To quantitatively characterize the changes in the signal

from the vessels, we calculated the average of the pixel ampli-
tude over the region indicated by the dashed box. The back-
ground signal was calculated by averaging over a region of
the same size but without vessels inside. The net signal from
the blood vessels is shown as a function of time in Fig. 4(b).
As can be seen, the average signal amplitude dropped quickly
by 60% in the first 100 s, and then increased slowly to 65% of
the initial value in the next 220 s. The initial drop in the signal
was probably due to a quick wave of injected saline before it was
homogeneously mixed with blood, while the slow increase later
was due to the diluting effect of blood.24
To demonstrate the flexibility of the handheld probe in clini-
Fig. 3 Handheld OR-PAM of vessels in a mouse ear. cal applications, we tested the system by imaging the human
skin. All the human experiments followed a protocol approved
by the Institutional Review Board administered by the Human
window, and an image with an SNR of 28 dB was obtained after Research Protection Office at Washington University in
averaging over 25 volumes, as shown in Fig. 3. St. Louis. We first imaged the blood vessels under a cuticle.
To demonstrate the fast imaging capability of the handheld To obtain higher SNR, the region of interest was scanned repeat-
OR-PAM system, the vessels in a mouse ear were continuously edly at a volumetric imaging rate of 2 Hz, and images were aver-
scanned after a tail vein injection of 0.6 mL of 0.9% saline. The aged over 20 C-scans. It should be noted that the small shift of
postinjection changes in the PA signal from the vessels were the same imaging region during the DAQ was corrected by
monitored with a temporal resolution of 2 Hz. Representative image translation transform, with the shifts calculated from
images taken at different times after the injection [Fig. 4(a)] image correlation. In Fig. 5(a), the vessels under the cuticle
show a decrease in the PA signal followed by a slower recovery can be seen clearly, with an SNR of 26 dB.

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 Lin et al.: Handheld optical-resolution photoacoustic microscopy

Fig. 5 OR-PAM of the human skin by the handheld probe. (a) OR-PAM image of capillaries in a cuticle.
(b) Photograph of the OR-PAM handheld probe imaging a red mole on a volunteer’s leg. (c) Photograph
of the mole. (d) OR-PAM image of the mole. The color bar shows the normalized PA signal amplitude.

The key advantage of a handheld system is its capability of and National Science Foundation (NSF) (1255930). The authors
imaging an area that is usually not accessible by a benchtop thank Professor James Ballard for his close reading of the
device. To demonstrate this advantage, a red mole on a healthy manuscript. L. V. Wang has a financial interest in
volunteer’s leg was imaged by the handheld OR-PAM probe, as Microphotoacoustics, Inc., which, however, did not support
shown in Figs. 5(b)–5(d). The scan fully covered the mole, and this work.
a single C-scan PAM image was able to resolve the features,
with an SNR of 20 dB. The optical fluence at the skin surface
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Journal of Biomedical Optics 041002-4 April 2017 • Vol. 22(4)

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 Lin et al.: Handheld optical-resolution photoacoustic microscopy

13. M. Chatni et al., “Functional photoacoustic microscopy of pH,” currently pursuing the Ph. D. in the Department of Electrical and
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20. S. Xu, C. Huang, and J. Zou, “Microfabricated water-immersible lage of Dr. Lihong Wang at Washington University. His current
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080505 (2012). under the tutelage of Prof. Lihong V. Wang. He is currently a postdoc-
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dilution on blood and plasma. Relation to the mechanism of coagulation Lidai Wang received the bachelor and master degrees from the
of normal and hemophilic blood,” Blood 6, 720–739 (1951). Tsinghua University, and received the PhD degree from the
25. American National Standards Institute, American National Standard for University of Toronto. After working as a postdoctoral research fellow
the Safe Use of Lasers, American National Standards Institute, New in the Washington University in St. Louis, he joined City University of
York (2000). Hong Kong in 2015. His research focuses on biophotonics, biomedi-
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saturation,” J. Biomed. Opt. 21, 061002 (2015). medical applications. He has published 30 articles in peer-
reviewed journals and has received four best paper awards from
Li Lin is currently a PhD student at Washington University in St. Louis, international conferences.
under the tutelage of Dr. Lihong V. Wang. He earned his master’s
degree at the University of Pennsylvania in 2013 and a bachelor’s Jun Zou received his PhD degree in electrical engineering from the
degree at Tianjin University in 2011. His research focuses on photo- University of Illinois at Urbana-Champaign in 2002. In 2004, he joined
acoustic tomography and microscopy. in the department of electrical and computer engineering at Texas
A&M University, where he is currently an associate professor. His cur-
Pengfei Zhang obtained his PhD in optics in 2008 from Shanghai rent research interests lie in the development of micro and nano opto-
Institute of Optics and Fine Mechanics. In 2009 he joined East electro-mechanical devices and systems for biomedical imaging and
Carolina University as a postdoc and worked on laser tweezers sensing applications.
and Raman spectroscopy. In 2012, he joined Los Alamos National
Laboratory as a postdoctoral research associate and his research Lihong V. Wang received his PhD degree at Rice University,
focused on advanced light microscopy and single molecule spectros- Houston, Texas. Currently, he is holding the Gene K. Beare distin-
copy. He joined Washington University in St. Louis in 2016 as a guished professorship of biomedical engineering at Washington
research associate and is working on photoacoustic imaging. University in St. Louis. He has published 400 peer-reviewed journal
articles and delivered 400 keynote, plenary, or invited talks. His goo-
Song Xu received his B.S. in optical and electrical engineering from gle scholar h-index and citations have reached 92 and over 34,000,
Huazhong Univ. of Science and Technology, P. R. China (2012). He is respectively.

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