IEEE TRANSACTIONS ON MEDICAL IMAGING, VOL. 33, NO.
2, FEBRUARY 2014
24-MHz Scanner for Optoacoustic Imaging
of Skin and Burn
Laetitia Vionnet, Jérôme Gateau, Mathias Schwarz, Andreas Buehler, Volodymir Ermolayev, and
Vasilis Ntziachristos*
Abstract—Optoacoustic (photoacoustic) imaging uniquely vi-
sualizes optical contrast in high resolution and comes with very
attractive characteristics for clinical imaging applications. In
this paper, we showcase the performance of a scanner based on
a 24 MHz center-frequency 128 element array, developed for
applications in dermatology. We perform system characterization
to examine the imaging performance achieved. We then showcase
its imaging ability on healthy tissue and cancer. Finally, we image
burns and human lesions in vivo and gain insights on the benefits
and challenges of this approach as it is considered for diagnostic
and treatment follow-up applications in dermatology and beyond.
Index Terms—Animal models, burn, dermatology, imaging,
optoacoustic, photoacoustic, skin.
I. INTRODUCTION
O PTOACOUSTIC tomography has demonstrated optical
contrast imaging through entire small animals [1] or
human tissues [2] in high resolution. Implementations in the
sub-10 MHz ultrasonic region can achieve penetration depths
of several centimeters with resolutions ranging in the few
hundreds of micrometers. This performance enables structural
and functional optical imaging of tissues at several millimeters
to centimeters depth [3]–[5]. Endogenous tissue absorbers such
as hemoglobin allow visualization of anatomical structures like
vasculature [2], [6] or physiological parameters, for example
tissue oxygenation and hypoxia [7]. The use of exogenous
photo-absorbing agents is also increasingly considered to stain
tissue biomarkers in vivo and reveal cellular or sub-cellular
processes [4]. Photo-absorbing agents have been also used to
image dynamic physiological processes such as tissue perfusion
[8]–[10].
Manuscript received September 30, 2013; accepted October 23, 2013. Date
of publication November 07, 2013; date of current version January 30, 2014.
This work was supported by the ERC Advanced Investigator Award N 233161-
MSOT. The work of A. Buehler and V. Ntziachristos was supported by the Eu-
ropean Union project FAMOS (FP7 ICT, Ccontract 317744). Asterisk indicates
corresponding author.
This paper has supplementary downloadable material available at http://iee-
explore.ieee.org, provided by the authors.
L. Vionnet, J. Gâteau, M. Schwarz, A. Buehler, and V. Ermolayev are with
the Chair for Biological Imaging, Technische Universität München, 81675 Mu-
nich, and also with the Institute for Biological and Medical Imaging, Helmholtz
Zentrum München, 85764 Neuherberg, Germany.
*V. Ntziachristos is with the Chair for Biological Imaging, Technische Uni-
versität München, 81675 Munich, Germany, and also with the Institute for Bi-
ological and Medical Imaging, Helmholtz Zentrum München, 85764 Neuher-
berg, Germany (e-mail: v.ntziachristos@tum.de).
Color versions of one or more of the figures in this paper are available online
at http://ieeexplore.ieee.org.
Digital Object Identifier 10.1109/TMI.2013.2289930
0278-0062 © 2013 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission.
See http://www.ieee.org/publications_standards/publications/rights/index.html for more information.
535
Imaging using optoacoustics would be ideal for derma-
tology and more generally subcutaneous imaging applications.
Melanin, hemoglobin, abnormal vascular patterns, and several
other forms of optical contrast could be detected to resolve
skin conditions such as cancer, inflammation, auto-immune
diseases, or burns. Correspondingly, the method should be
designed to the skin dimensions and offer fast acquisition of
images to yield optimal visualization of skin conditions in
clinical settings. The optoacoustic imaging performance, e.g.,
the image quality or the imaging frame rate achieved, are highly
dependent on the spatio-temporal response of the detectors, the
tomographic detection geometry, and the scanning strategy.
Typically, resolution and depth can be exchanged, i.e., reso-
lution improvement associates with smaller penetration depth
[1]. In response, different optoacoustic implementations have
been reported, adapted to operate at different spatial settings
ranging from penetration depths of several centimeters [11],
[12], whole-body small animal imaging through 1–2 cm [6],
[8], [13], [14] or imaging of smaller biological structures such
as fish [15] or vasculature at higher resolution [3], [16].
Similarly to the resolution achieved in ultrasound, the optoa-
coustic imaging resolution depends on the frequency of the de-
tector employed. However, a difference between ultrasound and
optoacoustic imaging is that the latter generates broad ultrasonic
frequency responses from tissues, ranging from the sub-MHz
to several tens of MHz or higher. Such broadband response
is present in all optoacoustic measurements, whereas in ultra-
sound, the only acoustic frequencies present in tissue are those
emitted by the ultrasonic transducer or harmonics induced by
nonlinear propagation. The frequency content of optoacoustic
signals depends on the duration of the laser pulse and the size,
shape, and orientation with regards to the detector of the ab-
sorbing structures imaged [17]. For dermatology and subcuta-
neous imaging, capturing high-frequency components will be
essential for high resolution imaging.
Generally, the spatial response of a detector at high frequency
results to a trade-off between sensitivity and directivity. Fo-
cused detectors attain typically a large active surface yielding
high sensitivity but a narrow directivity pattern [18], especially
at higher frequencies. Conversely, small surface detectors have
a broader directivity pattern but lower sensitivity. Although high
sensitivity would be desirable for high frequencies, it implies the
use of large-surface detectors which are not suitable for multi-el-
ement arrays, as it would be practical for fast or handheld scan-
ning schemes. Instead, use of 1-D ultrasound arrays, in analogy
to clinical ultrasound, can create real-time images but forces the
use of low surface area detectors which can however be large
and focused in one direction [19]–[23].
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Fig. 1. Experimental setup for a tomographic acquisition performed in vivo on a mouse: (a) schematic drawing and (b) corresponding picture. The heat lamp and
the mask were used for the animal anesthesia, the other labeled components correspond to the optoacoustic imaging setup. A coordinate system was defined: the
x-axis corresponds to the axis of the linear detector array and the z-axis to the translation direction of the motor. The z-axis defines the elevation direction of the
system. (c) Schematic side view of the setup showing the reconstruction parameter L, the synthetic aperture length, used when reconstructing volumetric images
in the 3-D continuous acquisition mode.
Volumetric acquisition can be performed in several detection
geometries for tomographic acquisition, by combining rotation
and translation. Among all, horizontal translation of an array,
i.e., along a plane, is well suited for in vivo optoacoustic imaging
as it allows imaging independently of the size of the imaged
body part and its skin topology. The imaging performance of
planar scanning depends on the density of the detectors em-
ployed. For example coarse scanning may lead to discretization
artifacts (grating lobes) when the spatial frequencies in the target
image are not adequately sampled. Naturally, high spatial sam-
pling leads to longer acquisition times.
In the present paper, we consider the problem of high-fre-
quency linear arrays for applications in optoacoustic imaging
based on parallelized detection allowing large detector aperture
and dense spatial sampling along the array axis. The construc-
tion of high-frequency linear arrays poses design challenges re-
lated to the small dimensions and thicknesses needed for high
frequencies [23]. A linear array system at 30 MHz has been pre-
viously employed to visualize subcutaneous vasculature in rats
[24], the beating heart of young atymic nude mice [25], dye ac-
cumulation and clearance in sentinel lymph nodes [26] and the
pulsatile motion of blood vessels [27]. However, the array used
in these studies had only 48 elements, a small detection aper-
ture and a pitch around twice the ultrasonic wavelength at the
center frequency leading to a coarse spatial sampling and direc-
tive detection. Moreover, only eight elements were read out in
parallel impairing resolution in the scan direction due to motion
over multiplexed data. The laser wavelength range from 523 to
610 nm allowed for a penetration depth of only a few millime-
ters due to optical absorption.
Herein, we interrogate the implementation of a real-time
imaging system for dermatology applications. For this purpose,
we considered a 128 element array with a center frequency of
24 MHz and high density of elements (pitch of 70 m), con-
nected to a custom-built 128 high-frequency data acquisition
system, and mounted on a micro-positioning linear stage for
fine adjustment. We examined the system with excitation in the
near-infrared so that light absorption does not impose strong
signal attenuation with depth as compared to using visible
wavelengths and could penetrate several millimeters in tissue
as required for some skin conditions or the characterization of
burns. We interrogated the performance of volumetric imaging
IEEE TRANSACTIONS ON MEDICAL IMAGING, VOL. 33, NO. 2, FEBRUARY 2014
by translating the array in the elevation direction and examined
its performance to image anatomical structures in phantoms
and tissues in vivo. Perfusion of subcutaneous tumors was
also studied as a dynamic process using intravascular injection
of gold nanorods. Finally, following the assessment of the
imaging parameters of the system, we consider its performance
for dermatology applications. Findings in resolving burns and
skin lesions are discussed in the context of the suitability of
such scanner for clinical translation.
II. MATERIALS AND METHODS
A. Experimental Set-Up
The optoacoustic setup used in this study is shown in Fig. 1.
A tunable (690–900 nm) optical parametric oscillator laser
(Phocus II, Optotek Inc., Carlsbad, CA, USA) delivering
ns duration pulses with a pulse repetition frequency
(PRF) of 10 Hz and a fiber bundle with two line-shaped arms
(CeramOptecGmbH, Bonn, Germany) were used to generate
and guide the excitation light to the sample. A high-frequency
ultrasound linear array (LA28.0/128, Vermon, Tours, France)
comprising 128 elements, with an averaged center frequency
of 24 MHz and an averaged two-way (pulse echo -6 dB)
bandwidth of 60%, was used for detection of the optoacoustic
waves. The elements of the array had a height of 1.5 mm and
were cylindrically focused at a focal distance of 7.5 mm using
an acoustic lens, placed in front of the elements during the
manufacturing of the array [23]. The pitch of the array was 70
m with elements of 55 m width. The fiber-bundle arms were
positioned on each side of the multi-element linear detector and
oriented towards the focal line of the elements. In this config-
uration, the light fluence on the sample surface—about 4 mm
from the fiber output—was measured to be below the maximal
permissible exposure [28] i.e., 20 mJ/cm2. Data acquisition was
performed with a custom-made 128-channel data acquisition
system (DAQ) at a sampling rate of 125 MSamples/s and with a
12-bit resolution over a 16 mV range. The DAQ was triggered
by the laser and data from all the elements of the detector array
were acquired simultaneously at the PRF of the laser. The
fiber-bundle arms and the ultrasound array were fixed together
and the assembly was mounted on a high-accuracy translation
stage (M-605, 2DD, Physik Instrumente, Germany) to scan the
VIONNET et al.: 24-MHZ SCANNER FOR OPTOACOUSTIC IMAGING OF SKIN AND BURN
sample along the z-direction. Acoustic coupling between the
detector and the sample was done with transparent acoustic
gel. Because the detector was translated through the acoustic
gel, a large amount of gel was used to obtain efficient acoustic
coupling for all the positions and to ensure that several scans
could be performed successively without the need to reapply
gel [Fig. 1(a)]. Both the DAQ and the translation motorized
stage were computer controlled. The sample was placed on a
mobile platform to adjust its position along the x-axis and the
y-axis before measurements.
B. Acquisition Modes and Image Reconstruction
The system was used to generate both 2-D and 3-D optoa-
coustic images. Multiple frames were recorded for each optoa-
coustic image, and three different data acquisition modes were
used in this study: 1) multiple frame acquisition at a single posi-
tion of the illumination-detection assembly named 2-D dynamic
acquisition mode, 2) stepped linear scan of the illumination-de-
tection assembly along the z-axis with averaged acquisitions of
the ultrasound field at each discrete position named 3-D aver-
aged (3Da) acquisition mode, 3) continuous linear translation of
the illumination-detection assembly with nonaveraged acquisi-
tions at the PRF of the laser named 3-D continuous (3Dc) ac-
quisition mode. The first acquisition mode was used to generate
cross-sectional images of tissues and record dynamic processes,
the two others to obtain volumetric images of the samples. For
all modes, data were saved and image reconstruction was per-
formed after the acquisition.
1) 2-D Dynamic Acquisition Mode: For the 2-D dynamic ac-
quisition mode, the illumination-detection assembly was kept
fixed above the cross section of interest of the sample and frames
were acquired at the PRF of the laser (10 Hz) for 100 s. Acquired
signals were filtered with a third-order Butterworth bandpass
filter between 2 and 40 MHz, and a filtered backprojection al-
gorithm [29] was used to reconstruct the 2-D images with pixel
size of 12 m 12 m. To improve the signal-to-noise ratio
(SNR) of the images without losing any time point, a simple
rolling average was performed on 11 successive acquisitions. A
total of 995 individual images were reconstructed for the entire
acquisition.
The image contains nonphysical negative pixel values, which
is a direct consequence of using a conventional backprojection
algorithm. To improve visualization of 2-D images, the images
were normalized to their maximum and displayed between
and 1.
2) 3-D Averaged (3Da) Acquisition Mode: For the 3-D av-
eraged acquisition mode, the illumination-detection assembly
was moved by 150 m steps along the z-axis. This step size
corresponds to approximately half of the full-width half-max-
imum (FWHM) of the sensitivity field at the center frequency
of the array calculated for the fixed cylindrical focus [18]. For
each discrete position, the measured signals were averaged over
31 successive acquisitions before being saved. For each posi-
tion, a 2-D frame (x-y plane) of the 3-D optoacoustic image
was reconstructed using the averaged signals. Data were filtered
with a third-order Butterworth bandpass filter between 2 and
40 MHz, and images with pixel size of 12 m 12 m were
reconstructed using a filtered backprojection algorithm. Each
537
2-D frame was assumed to correspond to a cross section of the
sample perpendicular to the scan direction. The 2-D frames from
the entire scan were then stacked to form a 3-D image with a
spacing of 150 m. In this mode, the linear acquisition speed
along the z-axis was: 2.6 mm/min.
3) 3-D Continuous (3Dc) Acquisition Mode: For the 3-D
continuous acquisition mode, the illumination-detection as-
sembly was translated continuously at a speed of 50 m/s, and
signals were acquired continuously at the PRF of the laser. The
translation motion between two successive acquisitions was
then equal to 5 m. The Doppler effect induced by the motion
of the array during the acquisition, and the displacement of
the array during the optoacoustic wave propagation can be
considered as negligible at this translation speed, for the image
depth, and for the ultrasound frequencies considered here [30].
Because of the broad illumination of the sample, the optoa-
coustic sources were considered constant during successive
acquisitions. The 2-D frames (xy plane) of the 3-D optoacoustic
image were reconstructed by taking into account signals from
several acquisitions. Fig. 1(c) shows that for a slice located at
, data acquired in the interval —L
being the synthetic-aperture length in the direction of the array’s
translation—were used for the reconstruction using a filtered
backprojection algorithm. 2-D images were reconstructed every
48 m, i.e., 4 times the voxel size in the other directions as the
resolution along the z-axis is expected to be lower due to the
limited detection angle induced by the acoustic lens. Different
synthetic-aperture lengths L, were tested and the influence of
this parameter on the resolution and the SNR of the volumetric
image was evaluated. In this mode, the linear acquisition speed
along the z-axis was of 3 mm/min.
Volumetric images result from the 3Da and 3Dc acquisition
mode. When m the number of laser pulses necessary
for the acquisition of a 2-D frame of the volumetric image is
the same for the two modes. One difference was that in the 3Da
mode signals were time averaged while in the 3Dc mode they
were spatially averaged.
For the visualization of the volumetric images, maximal am-
plitude projection (MAP) images were computed. A threshold
was applied to the images using the histogram of the volumetric
image: all voxels with negative values were discarded because
of their nonphysical meaning and so were the first 2.7‰ voxels
with positive values considered as noise and the last 1‰ of the
voxels were saturated. The MAP images were interpolated to
a grid 10 times denser in the case of the measurement of the
resolution.
C. Tissue Mimicking Phantoms
Two tissue-mimicking phantoms were used to characterize
the capabilities of the system in terms of volumetric imaging at
mesoscopic scale. Optically absorbing objects were embedded
in turbid agar gels made of 2% w/v agar (Sigma, Germany)
and 6% v/v intralipid (Sigma-Aldrich, Germany). Phantom 1
consisted of 10- m-diameter black microspheres (Carboxylated
Polystyrene Microparticles, Polysciences Inc., Warrington, PA,
USA) and was used to compare the 3-D resolution and the SNR
of the volumetric images in 3Da and 3Dc acquisition modes.
Phantom 2 consisted of a cross made of two 10- m-diameter
538
black suture threads (Braun, Dafilon, Polyamidmonofil, Aes-
culap AG) and was used to show the ability of the system to
image complex and controlled structures. The optical excitation
wavelength when imaging the phantoms was set to 700 nm.
D. In Vivo Experiments
The in vivo imaging capability of the system was demon-
strated on two mice, a three-week-old healthy CD-1 mouse
(Charles River Laboratories, Sulzfeld, Germany), and a CD-1
nu/nu mouse (Charles River Laboratories, Sulzfeld, Germany)
with a subcutaneous tumor implanted in the lower back. Hair
in the imaged regions was removed with a hair removing lotion
(Veet Hair Removal Cream, Rechitt Benckiser, Germany).
During the experiment, the mice were positioned in the prone
position and anesthetized for the course of the experiment with
a mixture of isoflurane (2% v/v) and oxygen (Fig. 1). The body
temperature of the mice was maintained with a heat lamp.
Imaging of the CD-1 mouse (mouse 1) leg was performed
targeting endogenous contrast (hemoglobin) at an optical exci-
tation of 760 nm. For the CD-1 nu/nu mouse (mouse 2), a sub-
cutaneous tumor was imaged, 10 days after the subcutaneous
inoculation of 4T1 breast tumor cells (mammary gland
tumor, LGC standards, Wesel, Germany). Three different acqui-
sitions were performed successively on mouse 2 over 24 h. First,
the endogenous absorbers of the tumorous tissue were imaged
in 3-D with an optical excitation at 800 nm. Second, methylated
goldnanorods with a peak absorption at 800 nm (5e12 gold-
nanorods in 100 L, Nanopartz Inc., Loveland, CO, US) were
injected intravenously via a tail catheter. Optoacoustic images
were acquired during the injection using 2-D dynamic acquisi-
tion mode. Third, the tumor was imaged in 3-D, 20 min after the
injection of the contrast agent, and once more 24 h later. The last
two steps aimed at visualizing the extravasation of the contrast
agent in the leaky vasculature of the tumorous tissue.
After the in vivo measurements, the mice were euthanized
and then frozen. The bodies were then sliced with a Leica
CM1950 Cryostat and color images of the different slices were
taken. Mouse care, handling, and euthanasia were performed
according to the institutional and Bavarian government regula-
tion in the frame of approved animal protocol.
E. Imaging of Burns and Human Lesions
We further examined the performance of the scanner to image
skin conditions. Due to the high resolution and penetration depth
achieved by the scanner, we hypothesized that it would be well
suited for burns. For this reason we obtained fresh chicken legs
from the local grocery store and generated various skin lesions
using an iron rod heated to 450 C. We were particularly inter-
ested to examine whether the scanner could resolve the outline
of burns and therefore adjusted the time of iron contact to the
skin to induce superficial and deeper burns. The data was ac-
quired using the 3-D continuous acquisition (3Dc) mode over a
synthetic length of 150 m, in analogy to the mouse measure-
ments. The imaging wavelength was 700 nm.
To further examine the performance of the scanner in imaging
the human skin we imaged several benign lesions on healthy
volunteers. For human imaging a spacer was constructed that
held the focal point of the transducers approximately 1 mm
IEEE TRANSACTIONS ON MEDICAL IMAGING, VOL. 33, NO. 2, FEBRUARY 2014
under the tissue surface so that lesions protruding from the
skin and subsurface contrast could be visualized. The spacer
was built out of agar gel and was secured in place in front
of the transducer with a thin plastic membrane. Ultrasound
gel was further employed for acoustic coupling. The imaging
wavelength was 700 nm and data was acquired with the 2-D
dynamic acquisition mode.
III. RESULTS
A. Phantom Experiments
1) 3-D Resolution: The 3-D resolution of the system was
investigated by imaging 10- m-diameter photo-absorbing mi-
crospheres, whose optoacoustic peak emission frequency is ex-
pected to be greater than the highest recorded frequency [31].
Given that the resolution depends on the scanning and recon-
struction parameters, the resolution achieved was studied with
both the 3Da and 3Dc acquisition modes over a scan length of 10
mm. Fig. 2(a)–(f) presents the reconstruction of a 10- m-diam-
eter microsphere located around the focal distance of the array
along the y-axis and the center of the array along the x-axis for a
3Dc acquisition with a synthetic-aperture length of m
[Fig. 1(c)]. The MAP images along the three axes of the coor-
dinate system [Fig. 2(a)–(c)] show that despite the geometrical
symmetry of the microsphere, its reconstruction appears elon-
gated, especially along the z-direction. The FWHM dimensions
of the reconstructed microsphere were 0.29 mm in the elevation
direction [Fig. 2(d)], 97 m in the lateral direction [Fig. 2(e)],
and 22 m in the axial direction [Fig. 2(f)]. The FWHM di-
mensions were measured here on the MAP images to evaluate
the size of the reconstruction spot. Because of the small size
of the microsphere in comparison to the captured ultrasound
wavelength, these FWHMs also provide a good estimation of
the maximum achievable resolution. The discrepancy between
the FWHM values in the different directions is explained by
limited view issues [3]. The detection aperture is limited along
the lateral dimension (x-axis) by the finite size of the array, and
along the elevation direction (z-axis) by the fixed focus of the
array and the synthetic-aperture length. In the axial dimension
(y-axis), the maximal resolution depends mostly on the band-
width of the detector and is therefore not diffraction limited. Be-
sides, one can notice two peaks [Fig. 2(f)] which are attributed
to the electrical impulse response of the array’s elements.
In order to study the influence of the acquisition mode
(3Da or 3Dc) on the 3-D image quality the microsphere was
reconstructed for both acquisition modes. In addition, the
performance of the 3Dc acquisition mode was also studied
as a function of the synthetic-aperture length L–L ranging
from 10 to 450 m, increasing in 10- m steps. The FWHM
dimensions of the reconstructed microsphere were computed
along the three axes along with the signal-to-noise ratio (SNR)
of the image for all the reconstructions studied. The SNR
was defined here as the ratio between the maximum value in
the reconstructed microsphere and the standard deviation of
the background noise computed over voxels selected to
be away from the microsphere’s signal. The axial and lateral
resolutions appeared not to be affected by the acquisition
mode or the synthetic-aperture length L employed. Conversely,
VIONNET et al.: 24-MHZ SCANNER FOR OPTOACOUSTIC IMAGING OF SKIN AND BURN 539

Fig. 2. Image of a microsphere from phantom 1 for 3Dc acquisition mode. The 10- m-diameter microsphere was located around the focal distance of the detector
array along the y-axis and at the center of the array along the x-axis. The center of the coordinate system was chosen here as the center of the sphere. All images
shown were normalized to their maximum. Images (a)–(c) are MAP images along the y-axis, the z-axis, and the x-axis, respectively, produced from a volumetric
reconstruction performed with a synthetic-aperture length m. (d) Amplitude profile of (a) for the line . (e) Amplitude profile of (b) for the line
. (f) Amplitude profile of (c) for the line . FWHM shown in (d) represents the elevation resolution. The dotted lines superimposed on (a)–(c) indicate
the direction of amplitude profiles displayed in (d)–(f), respectively. (g) SNR (circle) and elevation resolution (cross) of the microsphere imaged as a function of
the synthetic-aperture length, L, used for the reconstruction. The solid horizontal lines indicate the values obtained after linear interpolation in the 3Da acquisition
mode.

the elevation resolution and the SNR were found to vary

with the acquisition mode and the synthetic-aperture length L
[Fig. 2(g)]. For the 3Dc acquisition mode, the SNR increased
with L. This increase was expected as in this mode, we average
over a number of acquisitions that increases with L and there-
fore the standard deviation of the background noise, appearing
in the denominator of the SNR, decreases with increasing L.
The elevation resolution depends on two factors. The first
factor is the size and geometry of the detector elements along the
z-direction, assuming that the dimension of each detector ele-
ment along the x-direction is much smaller. The second factor is
the synthetic aperture length which is composed from multiple
measurements along the z-direction. There is a slight improve-
ment in elevation resolution seen for synthetic aperture lengths
up to m in Fig. 2(g). However the elevation reso-
lution drops for lengthier synthetic apertures. This is because
the transducer employed herein is a cylindrically focused ul-
trasound array. Correspondingly, as the transducer moves away
from the plane of interest it loses sensitivity to signals from this
plane and distorts the corresponding optoacoustic signals that
are measured in the off-axes positions.
The synthetic-aperture length m is of particular in-
terest over other L values because the number of single acquisi-
tions used to reconstruct the image is the same as for the 3Da ac-
quisition mode. Inspection of Fig. 2(g) shows 0.32 mm elevation
resolution for the 3Da acquisition mode and 0.29 mm elevation
resolution for the 3Dc acquisition mode. Therefore we can con-
clude that the 3Dc acquisition mode is about 10% better than the
3Da acquisition mode. Fig. 2(g) also shows better image quality
for the continuous acquisition over other scan modes in terms
of SNR. Correspondingly, for all remaining volumetric acqui-
sitions shown, we selected the 3Dc acquisition mode, which is
faster than 3Da, and a synthetic-aperture length m 2) Imaging of Crossing Wires: Fig. 4 depicts images recon-
which showed optimal performance in terms of image quality.
Fig. 3 shows the MAP images of the reconstruction of
phantom 1 for the entire scan. The MAP images were normal-
ized and saturated at 40% of the maximum after thresholding.
Fig. 3. Reconstructions of phantom 1 for data acquisition in 3Dc mode with
a synthetic-aperture length of 150 m in the scan direction. (a) MAP image
along the x-axis. (b) MAP image along the z-axis. The superimposed rectangles
indicate the location of the sub-volume reconstructed in Fig. 2. Voxel values
were normalized and saturated at 40% of the maximum after thresholding.
The MAP image along the z-axis [Fig. 3(b)] illustrates the
presence of 10 m microspheres randomly spaced within the
reconstructed field of view of 9 mm 9 mm in the x and y
directions and 10 mm in z, the direction of the array’s transla-
tion. The y coordinate indicates the distance to the transducer
array and the x coordinate corresponds to the width of the array
( at the center of the array). In this view, the spheres can
be clearly distinguished and are reconstructed with a consistent
resolution in the lateral and axial dimensions independently
of the x and y coordinates [Fig. 3(a)]. The MAP images in
Fig. 3(b) show that, consistently with the reconstruction in
Fig. 2, the elevation resolution is lower than the lateral and
axial resolution, and that it degrades with depth (increasing
y coordinate) all over the translation range of the scan. The
degradation could be attributed to the decrease of the relative
aperture in the elevation direction with increasing y coordinate,
and to the smearing artifacts.
structed from phantom 2, i.e., the two 10- m-diameter black
threads arranged in a bent cross. The images shown were pro-
duced using the 3Dc acquisition mode. MAP images from the
volumetric reconstruction (Fig. 4 and video 1) shows that the
540 IEEE TRANSACTIONS ON MEDICAL IMAGING, VOL. 33, NO. 2, FEBRUARY 2014

Fig. 4. Reconstruction of phantom 2 (a cross made of 10- m-diameter thread)

Fig. 5. In vivo imaging of the left thigh of mouse 1. (a) Cross-sectional 2-D slice
for data acquisition in 3Dc mode and with a synthetic aperture length
from a data acquisition in 3Dc mode. Arrow 1 shows the thigh surface whereby
m. (a) MAP image of the entire region of interest along the y-axis. (b) and
the other arrows point to vascular and bone structures (b) Cryoslice color image
(c) 2-D frames corresponding to the superimposed lines on (a), and ordered by
in the same region of the thigh as in (a). Field-of-view of (a) is indicated in
decreasing z. For (a) the voxel values were threshold at 0.7 of the maximum in
(b) by a dashed rectangle. (c) MAP image along an axis belonging to the y-z
the image. Video 1 corresponds to Fig. 4(a) and shows rotating MAP images
plane, forming an angle of 38 with the z-axis. The scan length was 7 mm. The
around the z-axis of phantom 2. Video rate is of 10 fps. Two successive frames
coordinate system is superimposed in the top left corner, and a small green line
are separated by 2 angle.
on the left indicates the position of the slice (a) on the z-axis. Reconstructions
(a) and (c) were cropped to fit the calf region. Legend: 1-skin surface, 2-fibula,
3-vessel, 4-left ischiatic vein, 5-microvessels, 6-tibia. Video 2 corresponds to
arrangement of the two bent threads was accurately recovered. Fig. 5(c), and shows rotating MAP images around x axis at 10 fps with one
frame every 2 : starting angle: from the z axis, ending angle: .
Fig. 4(b) shows a slice in which the threads are well separated
and can easily be distinguished. Fig. 4(c) shows a slice close
to the location of the intersection of the threads, and where
the saddle point between the individual reconstructions of the
threads first develops. The distance between the two threads
at the saddle point corresponds to a resolution of 117 m in
the sense of the Sparrow resolution criterion [32]. This resolu-
tion performance is similar to the lateral resolution determined
with phantom 1. Similar to the microspheres, the reconstructed
threads appear elongated in the lateral dimension (x-axis) here
(Fig. 4(b)–(c) and video 1). The continuous and elongated shape
of the threads along the z-axis result in this case in an apparent
elongation in the lateral dimension especially in the bent por-
tions of the threads.

B. In Vivo Mouse Imaging

1) Thigh of a Healthy Mouse: Fig. 5(a) and (c) depicts the
reconstruction of the endogenous optical absorbers at 760 nm
in the right calf of mouse 1 from a 3Dc acquisition performed
in vivo and for a synthetic aperture length m. The
leg was positioned so that its proximal-distal axis was parallel
to the z-axis. Anatomical structures visible and labeled on the
cryoslice color image [Fig. 5(b)] could be recognized in the
corresponding 2-D slice [Fig. 5(a)] and in the MAP images
of the 3-D reconstruction (Fig. 5(c) and video 2). Besides the
large anatomical structures, micro-vessels, not visible on the
cryoslice image, could be visualized on the optoacoustic im-
ages [Fig. 5(a)]. The MAP images in Fig. 5(c) and video 2
show that continuous and branching microvasculature could be
reconstructed up to 5 mm deep below the skin. This implies
the appropriateness of the 24 MHz scanner to image subsurface
photo-absorbing structures in biological tissue in vivo and it is
consistent with the use of the 24-MHz scanner for dermatology
applications.
Fig. 6. Cross-sectional dynamic imaging of a subcutaneous tumor (mouse 2)
during the intravenous tail vein injection of gold nanorods in solution. Nanopar-
ticles were injected 40 s after the beginning of the acquisition over an injection
period of 20 s. (a) Reconstruction of the 2-D slice at s. Three re-
gions-of-interest (ROIs) are indicated with superimposed squares respectively
at the periphery and interior of the tumor and at interface with the host. Skin
surface and the interface with the host are enhanced with respectively dashed
and dotted white lines. (b) MAP profiles (along the x-axis) of the 2-D recon-
structions versus time. The three superimposed arrows indicate the y coordinate
of the center of each ROI. (c) Sum of the squares of the pixel values in the three
different ROI as a function of time. The superimposed dashed lines in (b) and
(c) indicate the beginning of the injection.
2) Subcutaneous Tumor: Perfusion Imaging by Injection of
Gold Nanorods: A cross-sectional image of the subcutaneous
tumor of mouse 2 was imaged in 2-D dynamic acquisition mode
during the intravenous injection of gold nanorods in solution
VIONNET et al.: 24-MHZ SCANNER FOR OPTOACOUSTIC IMAGING OF SKIN AND BURN 541

via the catheterized tail. Fig. 6(a) shows a reconstructed 2-D

slice revealing the tumor microvasculature in particular at the
periphery of the tumor below the skin and at the interface with
the host. The gold nanorods were injected 40 s after the begin-
ning of the data acquisition so as to provide a background level.
2-D slices were reconstructed for all time points, and the ampli-
tude profiles resulting from the maximum amplitude projection
of the 2-D slices along the x-axis were computed. These am-
plitude profiles are shown in Fig. 6(b) as a function of time. A
step increase of the amplitude can be noticed after the injection
(from time s), especially for y coordinates corresponding to
the periphery of the tumor, and the interface with the host. Fur-
thermore, the increase was assessed locally on three different
regions-of-interest (ROIs) [Fig. 6(a) and (c)]. The sum of the
squares of the pixel values for these three ROIs were computed
and presented in Fig. 6(c). A step increase of about 45% can
be noticed from the ROI including microvessels at the interface
with the host. This increase occurs with a few seconds delay,
due to the circulation time of blood in a mouse. Less significant
increases of respectively 10% and 6% occur for the ROIs at the
top and inside the tumor.
3) Subcutaneous Tumor: Volumetric Imaging: Volumetric
optoacoustic acquisitions were performed in 3Dc mode to
image the tumor of mouse 2 before and after the injection of the
gold nanorods. The injection itself was studied in Section III-B2
(Fig. 6). The tumor was imaged at three different time points:
just before the injection, approximately 20 min after the in-
jection, and 24 h after the injection. The time delay of 20
min between the injection and the second acquisition enabled Fig. 7. Images of the subcutaneous tumor of mouse 2 before injection, 20 min
homogenization of the gold nanorods in the blood volume. postinjection, and 24 h postinjection. (a), (c), and (e) MAP images along the
Comparing the MAP images along the z-axis before and 20 z-axis of the reconstructions corresponding to the entire scans (length 13 mm)
respectively before injection and, 20 min after and 24 h after injection. (b), (d),
min after injection [Fig. 7(a) and (c)], it can be noticed that the and (f) MAP images along the z-axis of a volumetric slice (2.4 mm thick along z)
contrast of the microvasculature at the skin surface and under in the core of the tumor. Images (a)–(f) were cropped to fit the tumor region, and
the tumor is enhanced by the presence of the gold nanorods. MAPs images (a)–(d) and (e)–(f) were saturated respectively at 60% and 70%
of their maximum value to appear of a similar contrast. (g) Picture of mouse
The location of the enhanced microvasculature is confirmed 2 before the experiment with the contour of the 2.4 mm volumetric slice in
by the comparison of MAP images of a volumetric slice in the white dashed line. (h) cryoslice color image of the tumor. The superimposed
core of the tumor [Fig. 7(d) and (f)]. The slice MAP images corners in images (a)–(f) and (h) delimit approximately the same area. Video 3
corresponds to Fig. 7 (c) and shows rotating MAP images around x-axis of the
show indeed that none of the microvessels that appeared after tumor 20 min after injection. The video rate is of 10 fps with one frame every
the injection in Fig. 7(c) is located in the core of the tumor. 2 : starting angle: 0 from the proximal-distal axis, ending angle: 90 .
Moreover video 3 shows with the 3-D reconstruction that few
structures are distinguishable inside the tumor 20 min after
injection. An amplitude increase at the tumor periphery was
detected after injection as shown already for a 2-D acquisition on chicken skin ex vivo is shown in Fig. 8(a). We see a superfi-
in Fig. 6. The distribution of the optical absorbers is different 24 cial “X” mark and a deeper single spot burn. Fig. 8(c) shows a
h postinjection [Fig. 7(e) and (f)]. In comparison with the other photograph of the cut through the spot-like burn along the white
time points, optical absorbers could be detected in the core of dashed line shown in Fig. 8(a) indicating a burn depth of approx-
the tumor. The thick slice MAP image [Fig. 7(f)] confirms that imately 4 mm. Tissue that was not affected by the burn displays
the new absorbers are located in the tissue volume and not at pink colour. Coagulated tissue had turned white and carbonized
the periphery as 20 min postinjection. These absorbers are most tissue brown became darker for increasing levels of carboniza-
likely extravasated gold nanorods that accumulated due to the tion. Fig. 8(b) shows a MAP of the optoacoustic data along the
enhanced permeability through the leaky vasculature of the y axis. Both the X-shape burn and the spot-like burn are visible
tumor. The area where the new absorbers appeared corresponds on the image with contrast coming from the burned tissue frag-
to the reddish core on the cryoslice image [Fig. 7(h)]. ments. Finally, Fig. 8(d) shows a MAP image along the z axis
of a 2-mm-thick slice through the spot-burn. The entire outline
C. Imaging of Skin of the burn, in all depths is clearly resolved. The optoacoustic
images shown in Fig. 8(b) and (d) were acquired from intact
1) Imaging Burns: The results from imaging burns are dis- tissue, i.e., before performing the cross-sectional cut shown in
played in Fig. 8. A photograph of the artificially induced burns Fig. 8(c).
542
Fig. 8. Results from imaging burns. (a) Photograph of two artificially induced
burns (a superficial “X” mark and a deeper single spot burn) on chicken skin ex
vivo. (c) Cross-sectional photography of the single spot burn along the dashed
line in (a) indicating that the depth of the burn extents to approximately 4 mm
below the surface. The cross-sectional cut was performed after the optoacoustic
images shown in (b) and (d) were acquired. (b) MAP of the optoacoustic data
along the y axis. Both burns display strong contrast to the surrounding tissue.
(d) MAP image along the z axis of a 2-mm-thick slice including the spot-burn.
The entire outline of the burn, in all depths is clearly resolved; indicating that
the scanner developed can be employed to examine depth of burns.
2) Imaging Human Lesions: Fig. 9 summarizes representa-
tive results obtained from benign lesions. Fig. 9(a) shows a pho-
tograph of a small inflamed lesion stemming from a mosquito
bite that had been scratched open in the center. The total size
of the inflammatory area around the mosquito bite measured
approximately 8 mm in diameter. Correspondingly Fig. 9(b)
shows an optoacoustic cross section through the lesion, whereas
Fig. 9(c) shows the image obtained from healthy skin 2 cm away
from the area scanned in Fig. 9(b). Similarly, Fig. 9(e) shows the
cross-sectional appearance of the small freckle seen on Fig. 9(d)
from the forearm of a volunteer. In this case the strong absorp-
tion of melanin makes the freckle clearly visible on the image
(arrow number 1) in comparison to the rest of the skin (arrow
number 2). The freckle measured less than a few hundred mi-
crons in depth. Other structures can also be seen in the image,
including the upper and lower boundary of the median vein of
the forearm, as indicated with arrow 3.
In both cases of imaging the burn and human skin lesions
there is an evident capacity to provide high-resolution optical
imaging within at least 4 mm inside the tissue.
IV. DISCUSSION
We examined in this study the performance of a 24-MHz
array detector for biomedical optoacoustic imaging of skin and
subcutaneous tissue in vivo. Emphasis was placed on operating
with a high frequency and high density conventional linear ul-
trasound array and electronics allowing simultaneous acquisi-
tion of all the detector elements of the array. This development
was considered in particular as it relates to imaging applica-
tions for dermatology. Therefore the capabilities of the imaging
system in terms of resolution, dynamic imaging performance
IEEE TRANSACTIONS ON MEDICAL IMAGING, VOL. 33, NO. 2, FEBRUARY 2014
Fig. 9. Imaging of human skin lesions. (a) Photograph of an inflamed mosquito
bite measuring approximately 8 mm in diameter. The lesion, scratched open in
the center, had the characteristic redness of inflamed tissue and was protruding
by a few millimeters above the surrounding skin. (b) Cross section through the
lesion obtained with the 24-MHz detector operated in the 2-D dynamic acqui-
sition mode, resolving a clearly elevated lesion; the elevation being less than
1 mm. This appearance is in clear difference to the cross-sectional appearance
of healthy skin, shown in (c). The cross section in (c) was obtained in the same
manner as in (b) from an area 2 cm below the lesion. (d) Photograph of a small
mole measuring approximately 800 m in diameter and (e) depth resolved op-
toacoustic image. 1-mole, 2-skin surface, 3-upper and lower boundary of a vein
passing below the mole as seen in the photograph from the bluish color.
and the overall ability to image subsurface structures and func-
tion was examined using phantoms, animal tissues in vivo and
ex vivo and human imaging in vivo. In addition, to examine the
future possibility of detecting administered contrast agents, the
ability to image tumor perfusion was also studied in vivo using
gold nanorods after their systemic administration in a mouse.
For operation in single-slice mode, real-time imaging at the
PRF of the laser and the use of a micro-positioning system al-
lowed fine positioning of the detector to a plane of interest. For
the tumor images presented in Fig. 6, a slice comprising sev-
eral vessels was chosen. Once chosen, the detector was kept
in position, and dynamic imaging of perfusion could be per-
formed. In this mode, imaging is performed on a thin volumetric
slice whose thickness corresponds to the elevation dimension of
the sensitivity pattern in the focused direction of the array. At
24 MHz for the array used in this study, the slice thickness is
on the order of 300 m, and a fine adjustable positioning is re-
quired for high quality imaging. Different signal increases were
observed at the base, the top and inside the tumor. The lower
relative increase of the pixel values in the center of the tumor in
comparison with the tumor periphery match observations made
with contrast-enhanced ultrasound in a previous study [33]. The
difference of signal increase between the base and the top of the
tumor could simply correspond for the imaged slice to a differ-
ence of vessel size. While our study demonstrate the possibility
of dynamic imaging during the injection of a contrast agent on
one specific acquisition, a specific study with several imaging
modality and multiple mice will be needed to confirm the trends
in terms of contrast wash-in in the tumor. Such a study is beyond
the scope of the paper.
We considered an alternative mode of acquisition for volu-
metric imaging. The purpose of this investigation was two-fold.
First, we wanted to examine the imaging performance as it re-
lates to tissue imaging because three dimensional imaging im-
proves visualization of continuous structures such as micro-ves-
VIONNET et al.: 24-MHZ SCANNER FOR OPTOACOUSTIC IMAGING OF SKIN AND BURN
sels. Second, we employed this mode to characterize the eleva-
tion resolution in association with the slice thickness achieved
by the focusing ability of the detector. For volumetric imaging,
the high spatial density of successive array positions along the
translation direction was shown as expected to result in better
elevation resolution and SNR than sparser spatial sampling and
time averaging. For the same number of acquired data, we found
that the continuous translation mode (3Dc acquisition mode)
yielded better image quality than the averaged mode (3Da ac-
quisition mode), even at reduced acquisition times by about 15%
due to completely utilizing the laser pulse excitation and not
dropping signals from any pulse. Compared to previous imple-
mentations [34], [35], the implementation of the 3Dc acquisi-
tion mode has several advantages owing to the higher sensitivity
and lateral detector element density that yields higher image
quality than previous detectors. Importantly, the ability to detect
in parallel from all 128 elements allowed for the first time true
real-time cross-sectional subcutaneous imaging operation and
faster scanning times at volumetric imaging operation. Never-
theless, motion-induced phase-differences between successive
acquisitions can potentially affect the image quality in terms of
resolution and SNR.
In the presented implementation, it took 1.1 s to acquire an
image in the 2-D mode with signals being averaged over 11 con-
secutive pulses. In the 3-D mode the acquisition time was in the
order of 1 min per 3 mm scan in the elevation direction. The
acquisition speed was primarily limited by the laser pulse rep-
etition frequency. The use of a different laser technology, such
as the one recently developed by our group [11], enabling high
power and fast tuning rate nanosecond pulsing of up to 100 Hz
in the near infrared, would significantly increase the scanning
speed. Higher scanning speed will also open new possibilities
for the observation of biological processes, better integration
with dermatology applications and would facilitate motion-free
multi-spectral imaging, insensitive to breathing motion or other
movement, compared to the images performed herein.
Because of the fixed cylindrical focus of the detectors in the
elevation direction, the resolution in this direction was approx-
imately an order of magnitude less than the axial resolution.
Also directional absorbers such as vessels were better visible
in planes parallel to the detection surface, as previously con-
firmed [36]. To keep the advantages of using linear ultrasound
arrays, a rotation motion of the array, either around the y-axis
or the x-axis [14], [36], can be introduced to synthetically in-
crease the detection angular aperture and therefore improve the
resolution and the partial view problem in the elevation direc-
tion. However, to avoid longer scanning time, the directionality
can simply be taken into account by choosing the orientation of
either the detection surface or the array if the main orientation
of the structures of interest is known. In addition, anisotropic
resolution is common in other imaging modalities as well; for
example in magnetic resonance imaging it is common that the
slice thickness is an order of magnitude worse than the in-plane
resolution when using plane-selection spin-echo acquisitions.
Regardless, with fast acquisitions, a few orientations could also
rapidly be tested once the animal under anesthesia or the human
body part is in position to select the most appropriate ones be-
fore any further imaging. To keep the elevation resolution con-
543
stant with depth for isotropic absorbers as the microspheres of
phantom 1, and then avoid the degradation with depth of the el-
evation resolution show on Fig. 3, the reconstruction algorithm
could be modified. Instead of using a single synthetic-aperture
length L optimized for the middle depth to reconstruct all depth,
a dynamic aperture reconstruction could be implemented by in-
creasing L with the depth. The choice of m in this
study corresponded to a good compromise in terms of SNR and
elevation resolution. It also corresponds to the elevation width
of the focal zone around the focus for the highest captured fre-
quencies [18]. With higher L, the SNR increases as the noise
decreases by incoherent summing, but the elevation resolution
degrades as lower frequencies are enhanced by coherent sum-
ming. A reconstruction that takes into account the spatio-tem-
poral impulse response of the detectors [37], [38] is expected to
further improve image resolution and quality as it takes into ac-
count the frequency dependent signal distortion due to the lens.
Such reconstruction algorithm are beyond the present study, but
will be investigated in the near future.
The extravasation of gold nanorods in solid tumors was also
studied to examine the ability to visualize dynamic events and
the effects of contrast enhancement. Volumetric images were ac-
quired before injection, 20 min and 24 h after the injection of the
nanoparticles; their extravasation resulting in the appearance of
increased absorption in the core of the tumor. This finding cor-
relates well with the observation of Li et al. with a different
optoacoustic system and gold nanoshells [39] on time scale of a
few hours (5.8 h). The type of gold particles, tumor and time of
observation differ from the present study but similar clustered
structures, heterogeneously distributed in the tumor, were ob-
served in both studies. Our system enabled imaging of larger
volumes, and an extended and systematic observation of the ex-
travasation of gold nanoparticles on a longer period of time,
compared to the previous study by Li et al. [39], and could be
an interesting tool also for studies in cancer biology. The tumor
perfusion could also be imaged at shorter time (tens of seconds),
and show different contrast enhancement depending on the loca-
tion and the presence of large microvessels. Such imaging con-
veys some physiological information at mesoscopic scale.
In the past decades several optical imaging techniques with
possible applications in dermatology have emerged. These in-
clude confocal imaging, multiphoton tomography and optical
coherence tomography (OCT) including OCT using Doppler
shift. With these techniques the epidermis, papillary dermis, and
superficial reticular dermis of healthy skin [40], the microvas-
culature of solid tumors [41], the retinal and choroidal vascu-
lature [42], hair follicles, blood vessels, sweat ducts, as well as
superficial skin conditions [43] have been imaged. These tech-
niques have demonstrated superior performance over visual in-
terrogation or photographic approaches. An important limita-
tion of these techniques however is that the accessible depth is
typically restricted to less than 1 mm. Consequently, melanoma
thickness or tumor invasion depth could not be generally deter-
mined [44] except in very superficial lesions of less than 1 mm
[43]. Melanoma thickness is an important factor in the prognosis
of recurrence and metastasis [45], [46]. In this context, the tech-
nique presented herein warrants its investigation for dermato-
logical applications, since it does not have such penetration limi-
544
tations. Fig. 8(d) reveals that images of burns penetrating at least
4–5 mm into biological tissue can be reliably reconstructed with
the 24-MHz linear array. These findings indicate that imaging
of burn depth could be possible at 24 MHz. Further interroga-
tions should be conducted to determine if the resolution offered
at 24 MHz would also be sufficient for visualizing malignant
melanoma penetrating several millimeters deep into skin tissue
or if adjustments in the frequency utilized may be required, at
the expense of penetration depth, to better visualize melanoma
invasion.
As a concluding note we would like to observe that while
dermatology is well served today by visual inspection and good
quality photography, there may be a particular segment of appli-
cations that can benefit not only by the ability to offer cross-sec-
tional images and assess the depth of different lesions but also
by combining the spectral and time characteristics of optoa-
coustic imaging to improve the detection specificity and better
understand the underlying physiology [7]. Future studies will
include more detailed human studies and statistics to better un-
derstand the technology application on particular diseases.
ACKNOWLEDGMENT
The authors would like to thank Dr. N. Bézières and
P. Symvoulidis from the Institute for Biological and Medical
Imaging for their support and the fruitful discussions. The
authors would also would like to thank S. Glasl and U. Klemm
for their technical support and the laboratory mouse handling.
All animal experiments were carried out as approved by the
district government of upper Bavaria.
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