J Med Ultrasonics (2014) 41:431–437
DOI 10.1007/s10396-014-0541-0

ORIGINAL ARTICLE

In vitro experiment using porcine artery for evaluationof ultrasonic measurement of arterial luminal surface profile
Yoshifumi Nagai • Magnus Cinthio •Hideyuki Hasegawa • Martin Bengtsson •Mikael Evander • John Albinsson • Hiroshi Kanai

Received: 28 January 2014 / Accepted: 26 March 2014 / Published online: 7 May 2014
Ó The Japan Society of Ultrasonics in Medicine 2014

Abstract
Introduction In early-stage atherosclerosis, the luminal
surface of the arterial wall becomes rough because of
detachment of endothelial cells and degeneration of theinternal elastic layer. Therefore, it would be useful if minuteluminal surface roughness of the carotid arterial wall, which
occurs in the early stage of atherosclerosis, could be measured noninvasively with ultrasound. The injured luminal
surface is believed to have roughness of a few hundred
micrometers. However, in conventional ultrasonography, theaxial resolution of a B-mode image depends on the ultrasonic wavelength (150 lm at ultrasonic center frequency of
10 MHz) because a B-mode image is constructed using theamplitude of the RF echo signal. Therefore, such surface
roughness cannot be measured accurately from a conventional B-mode image. Recently, we successfully measured
such minute surface profile transcutaneously using the phaseshift of an ultrasonic echo from the carotid arterial wall. In
our previous validation experiment, a silicone phantom withminute surface roughness of 10–20 lm was measured.
However, the feasibility of our proposed method has neverbeen validated using biological tissues.
Y. Nagai  H. Hasegawa  H. Kanai (&)
Graduate School of Biomedical Engineering, Tohoku University,
6-6-05 Aramaki-aza-Aoba, Aoba-ku, Sendai, Miyagi 980-8579,
Japane-mail: hkanai@ecei.tohoku.ac.jp
M. Cinthio  M. Bengtsson  M. Evander  J. Albinsson
Department of Biomedical Engineering, Faculty of Engineering,
Lund University, Lund, Sweden
H. Hasegawa  H. Kanai
Graduate School of Engineering, Tohoku University,
6-6-05 Aramaki-aza-Aoba, Aoba-ku, Sendai,
Miyagi 980-8579, Japan

Materials and methods In the present study, luminal
surface roughness of a porcine artery was measured and theresult was evaluated by comparing it with the result measured using a stylus profilometer.
Results and conclusion The root mean squared difference
between the surface roughness measured by ultrasound andthe stylus profilometer was 10.5 lm. This result proves thatour proposed method can be used to measure minute surface roughness of biological tissue.
Keywords Luminal surface of arterial wall  Roughness Porcine artery  Atherosclerosis

Introduction
Circulatory diseases are considered to be mainly caused byatherosclerosis, and early diagnosis or prevention of atherosclerosis is important. Medical ultrasound is widely used for
diagnosis and treatment in clinical settings. Diagnostic
ultrasound is noninvasive and, thus, can be repeatedly
employed. Such properties of diagnostic ultrasound aresuitable for diagnosis of atherosclerosis because the progressof atherosclerosis needs to be followed for a long term [1–4].
The luminal surface of a clinically healthy artery is covered by endothelial cells and is smooth. However, in the earlystage of atherosclerosis, endothelial cells and the internal
elastic layer are damaged, and endothelial cells are detachedfrom the luminal surface [5, 6]. As a result, the luminal
surface of the arterial wall will have roughness measuring a
few hundred micrometers [7–9]. Therefore, it would be
useful to measure such minute surface roughness of the
carotid arterial wall for early diagnosis of atherosclerosis.
The measurement of such minute surface roughness requires
a high spatial resolution. However, in conventional

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J Med Ultrasonics (2014) 41:431–437

Fig. 1 Illustration of
displacement of the arterial wall
between the 0-th frame and n-th
frame

ultrasonic diagnostic equipment, the spatial resolution in theaxial direction depends on the ultrasonic wavelength(150 lm at ultrasonic center frequency of 10 MHz) becausea B-mode image is constructed using the amplitude of theultrasonic RF echo. Arihara et al. [10] measured the micronorder surface profile of an object made of silicone rubber
using the phase shift of RF echoes during sweeping of an
ultrasonic beam. However, in this method, the luminal surface cannot be measured accurately in vivo because thesound speed in the propagation medium between the skinsurface and the carotid artery is inhomogeneous and theestimation of the distance from an ultrasonic probe to thesurface (corresponding to the surface profile) is significantly
affected by such inhomogeneity of the speed of sound.
In recent years, owing to significant improvements inthe performance of ultrasonic diagnostic equipment, themovement of the arterial wall not only in the arterial radial
direction but also in the longitudinal direction has come tobe observed in a conventional B-mode image [11]. This
longitudinal movement is considered to be caused bycontraction of the heart. Cinthio et al. showed that minuteluminal surface roughness could be measured using thearterial wall movement in the arterial longitudinal direction
without sweeping an ultrasonic beam [12, 13]. In thismethod, the phased-tracking and block-matching methods
were used to measure displacements of the luminal surface
caused by roughness and pulsation of the arterial diameter
during a cardiac cycle. In their validation experiments, thesurface profiles of silicone phantoms with surface roughness of 13–33 lm were measured, and minute surface
roughness was shown to be measured by ultrasound.
However, the movement of the arterial wall in the longitudinal direction is small (less than 1 mm) and, thus, thelength of a region measured by a fixed ultrasonic beam islimited. Therefore, Kitamura et al. [14] expanded themeasurable region by combining multiple surface profiles
measured by multiple ultrasonic beams.

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In our series of studies, our method was validated byphantoms made of silicone rubber and has never beenvalidated using biological tissues. Therefore, in the present
study, we measured a porcine artery in a water tank andevaluated our proposed method by comparing the results to
the results of measurement with a stylus profilometer.

Materials and methods
Measurement of surface roughness of arterial wall [14]Figure 1 illustrates the displacement of the arterial wall
during a cardiac cycle. In a fixed ultrasonic beam, the axial(z-axis) displacement would be detected when the luminal
surface is rough and the arterial wall moves in the arterial
longitudinal (lateral) direction. In addition, the arterial wall
also moves in the arterial radial (axial) direction owing to
expansion of the artery caused by an increment of bloodpressure during a cardiac cycle. The sum of these axial
displacements can be estimated by the ultrasonic phasedtracking method [15–17].
In Fig. 1, the lateral position of the m-th ultrasonic beam
is denoted by xm (xm = mdx, where dx is the spacing
between ultrasonic beams). The axial (z-axis) displacementof the posterior wall surface Ddðxm ; nÞ  Ddðm; nÞbetween the n-th frame [t (s)] and the (n ? 1)-th frame[(t ? DT) (s)] (DT frame interval) obtained by the phasedtracking method is expressed by the difference between theaxial positions zðxm ; nÞ  zðm; nÞ and zðxm ; n þ 1Þ zðm; n þ 1Þ of the surface in the n-th and (n ? 1)-th frames
as follows:Ddðm; nÞ ¼ zðm; n þ 1Þ  zðm; nÞ:ð1Þ

In the present study, the displacement d(m, n) was
measured by the phased-tracking method, and the mea^ nÞ contains both the global
sured displacement dðm;J Med Ultrasonics (2014) 41:431–437

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displacement of the arterial wall dg(n) due to expansion of
the artery and the displacement ds ðm; nÞ owing to surface
roughness, where ^ denotes the estimate. The measured^ nÞ is expressed as follows:
displacement dðm;^ nÞ ¼ dg ðnÞ þ ds ðm; nÞ:
dðm;

ð2Þ

The width of the measured region (about 8 mm) is muchsmaller than the wavelength of the pulse wave (several
meters at 1 Hz) propagating along the carotid artery.
Therefore, the global wall motion between the n-th and(n ? 1)-th frames at positions fxm g ðm ¼ 0; 1; 2; . . .; M  1Þis assumed to be the same. The axial global displacementdg ðnÞ caused by global wall motion can be estimated byaveraging the estimated axial instantaneous displacements^ nÞg as follows:fDdðm;d^g ðnÞ ¼

n1
X

Dd^g ðiÞ;ð3Þ

Fig. 2 Procedure for measurement of surface roughnessi¼0

Dd^g ðnÞ ¼

1
X
1M
^ nÞ;
Ddðm;
M m¼0

ð4Þ

where M is the number of ultrasonic beams.
By defining the lateral (arterial longitudinal) displacement measured in the m-th ultrasonic beam in the n-thframe by l(m, n), the lateral and axial positions of theluminal surface measured by the m-th ultrasonic beam in
the n-th frame are x = xm ? l(m, n) and z(m, n), respectively. Therefore, the height of the surface (= surface
profile) hm(x; hm0) measured by the m-th ultrasonic beam
can be obtained as a function of the lateral positionx = xm ? l(m, n) as follows:h^m ðx; hm0 Þ  h^m ðxm þ ^lðm; nÞ; hm0 Þ ¼ z^ðm; nÞnX
Dds ðm; iÞ;¼ hm0 þ

ð5Þ

i¼0

where
^ nÞ  Dd^g ðnÞ;Dd^s ðm; nÞ ¼ Ddðm;ð6Þ

and hm0 is the initial height.
The arterial longitudinal displacement l(m, n) needs tobe estimated by 2D tracking methods, e.g., block-matching
technique [18] with reconstructive interpolation [19], toobtain the surface profile hm(x; hm0) as a function of lateralposition x ¼ xm þ lðm; nÞ. Furthermore, surface profiles{hm(x; hm0)} were measured in multiple ultrasonic beams^ in a{m} and were combined to obtain a surface profile hðxÞwider region.
The above-mentioned procedure of measurement of
surface roughness of the carotid arterial wall is shown in
Fig. 2.

Evaluation of measured surface profile
In the present paper, the difference between the surface
profiles, e.g., surface profiles h^a ðxÞ and h^b ðxÞ, obtained
from different measurements was evaluated using the root
mean squared difference (RMSD) eRMSD defined asfollows:
vﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ
u K 1
u1 X 2
eRMSD ¼ t
ð7Þh^a ðxi Þ  h^b ðxi Þ ;K i¼0
where K is the number of positions where the surface
profile was measured.
Also, the root mean squared roughness (Rq) was used toshow the average roughness. The Rq is defined as follows:
vﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ
u K 1
u1 X ^ i Þ  h 2 ;Rq ¼ t
ð8Þ
hðx
K i¼0
^ i Þg.
where h is the average of fhðxIn vitro experiments
Experimental system
Figure 3a shows a healthy porcine descending aorta used inexperiments, and the aorta was cut opened in measurements, as shown in Fig. 3b. Figure 4 shows a picture of theluminal surface of the porcine artery observed with an
optical microscope (BX40, Olympus Corp., Japan). Measurements were conducted with an ultrasound system(ULA-OP, Firenze Univ., Italy) and a stylus profilometer

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Fig. 3 a Picture of the healthy
porcine descending aorta usedin the present study. b Picture of
the cut-open porcine descending
aorta without tissue fixation

were fixed using formalin. The concentration was 19 %,and the time for tissue fixation was about 8 h. After thetissue fixation, the porcine artery was fixed on a rubber
plate using needles. Also, markers (needles) specifying themeasured section were put on the luminal surface.
First, ultrasonic measurements were conducted. Porcine
arteries were measured in a water tank and were moved
using a movable stage, as shown in Figure 5, to simulate
the displacement of the carotid artery. The velocities in thelateral and axial directions were 1.4 and 0.7 mm/s,
respectively. The ultrasonic probe was fixed using a fixed
stand. After ultrasonic measurements, the porcine arterywas measured with a stylus profilometer. The resolution of
the stylus profilometer was 1.5 nm in the axial direction,and the stylus force was 5.0 mg (50 lN).
Fig. 4 Picture of the luminal surface of the porcine artery without
tissue fixation observed using an optical microscope

Fig. 5 Illustration of the experimental system. The porcine arterywas moved using a movable stage to simulate displacement of carotid
arterial wall

(XP-1, Ambios technology Inc., USA) to compare theresults obtained by these systems and validate the accuracyof the ultrasonic measurement.
In the measurements with the stylus profilometer, theluminal surface of the porcine artery might be deformed
due to pressure of the stylus. Therefore, to reduce the effectof pressure of the stylus, a pretreatment was applied to the
porcine artery before measurements. To increase thetoughness and to fix the surface profile, the porcine arteries

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Experimental results
Figure 6a shows a B-mode image of the porcine artery. The
RF data were acquired using a 9.38-MHz linear-type probe,
and the frame rate was set at 160 Hz. Figure 6b showssurface profiles measured by ultrasound. The measurements were performed three times to confirm the reproducibility of the proposed method. A minute surface profile
was found to be measured reproducibly by our proposed
method, as shown in Fig. 6b.
Figure 6c shows the surface profiles measured byultrasound (red line) and a stylus profilometer (blue line).
The surface profile measured by ultrasound in Fig. 6c is theaverage of the surface profiles in Fig. 6b.
In measurement using the stylus profilometer, the same
position of the porcine artery identified by markers wasmeasured. The surface profile measured by a stylus profilometer was also averaged for three times. As shown in
Fig. 6c, overall, the surface profile measured by ultrasoundagreed well with that measured by the profilometer, and theroot mean squared difference eRMSD between these two
profiles was 10.5 lm. Slightly larger errors around thelateral positions of 2–5 mm might be caused by the disagreement of the regions measured by ultrasound and the
profilometer because the width of the ultrasound beam of
0.5 mm (width at half maximum) was larger than the
diameter of the stylus of 2 lm.

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Fig. 6 a B-mode image of the
porcine artery. b Surface
profiles measured three times by
the proposed ultrasonic method.
c Averaged surface profile ofthe porcine artery measured by
the proposed method (red line)
and stylus profilometer (blueline)

In actual in vivo measurement, there is a medium with
non-uniform sound velocity distribution between the skinand the anterior wall of the carotid artery. To investigate
the effect of the propagation medium, the same porcine
artery was measured under the condition that a porcine
cutlet simulating the medium with non-uniform soundvelocity distribution was inserted between the porcine
artery and the ultrasonic probe. The thickness of the porcine cutlet was about 8 mm. Figure 7 shows the measured
surface profiles with (green line) and without (red line) the
porcine cutlet. Both surface profiles were measured byultrasound. RMSD between the two surface profiles was
6.2 lm. Although the difference was slightly larger aroundthe lateral position of 2 mm, the overall shapes of thesurface profiles measured with and without the porcine
cutlet were very similar to each other. From these results, it
was shown that minute surface roughness of several tens of

micrometers could be measured under a condition simulating in vivo measurement.

Discussion
We proposed an ultrasonic method for measurement ofminute surface roughness of the carotid arterial wall
occurring in the early stage of atherosclerosis. In our previous studies, the accuracy of our proposed method wasvalidated using phantoms made of silicone rubber, and thefeasibility of our method for the measurement of biologicaltissue had not been shown. Therefore, in the present study,
the accuracy of our proposed method was validated using
porcine arteries. To compare the surface profile measured
by ultrasound with that measured with a stylus profilometer, the porcine arteries were fixed with formalin to

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Fig. 7 Surface profiles of the
porcine artery measured with(green line) and without (redline) the porcine cutlet between
the porcine artery and ultrasonicprobe

Table 1 Root mean squaredroughness Rq of fresh porcine
arteries

Table 2 Root mean squaredroughness Rq of porcine arteries
with tissue fixation

Porcine artery

Rq (lm)Sample 118

Sample 2

14

Sample 3

22

Average18

Porcine artery

Rq (lm)Sample 1

23

Sample 2

20

Sample 3

17

Average20

increase the hardness because the stylus deformed thesurface profile when a fresh porcine artery was used.
However, the surface roughness of a porcine artery may bechanged by formalin fixation. Therefore, to examine theinfluence of tissue fixation on surface roughness, luminal
surface profiles of six arteries (three fresh, three fixed) fromthe same pig were measured by ultrasound. For evaluationof the change in surface roughness, root mean squaredroughness (Rq) was used. Tables 1 and 2 show the measured {Rq}. The average Rq of the fresh porcine arteries
was 18 lm, and that of the porcine arteries with tissue
fixation was 20 lm. The difference in {Rq} was 2 lm, andthis small difference indicates that the effect of tissue fixation can be ignored.

Conclusions
In the present study, the luminal surface profile of a porcine
descending aorta was measured by our proposed methods
and the result was evaluated by comparing it with the result
from measurement with a stylus profilometer. From theseresults, it was confirmed that the luminal surface profile,which has roughness of tens of micrometer, can be measured reproducibly and accurately by our proposed method.

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Furthermore, the medium with non-uniform sound velocity
distribution between the skin and anterior wall of the carotid artery was simulated by inserting a porcine cutlet
between the porcine artery and the ultrasonic probe. From
this measurement, it was also confirmed that the minuteluminal surface profile can be measured under the condition very similar to in vivo measurement.
Conflict of interest

None.

Ethical standard In this study, no laboratory animals were used;
only an excised porcine artery obtained from slaughterhouse waste
was used.

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