#2018 The Acoustical Society of Japan

Acoust. Sci. & Tech. 39, 3 (2018)

Compact test setup for sensitivity evaluation of photoacoustic contrast agent
Xi Zeng, Yosuke Mizuno, Kentaro Nakamura, Shinichiro Fuse and Hiroyuki Nakamura
Institute of Innovative Research, Tokyo Institute of Technology,
4259 Nagatsuta-cho, Midori-ku, Yokohama, 226–8503 Japan(Received 28 July 2017, Accepted for publication 25 September 2017)
Keywords: Photoacoustic system, Contrast agent, Evaluation, Sensitivity, Resonator
PACS number: 43.58.+z [doi:10.1250/ast.39.259]
1.

Introduction
Photoacoustic (PA) tomography has dramatically developed in the past 20 years and is expected to become acommon imaging method in hospitals in the next decade [1,2].
The ﬁrst generation of PA tomography adopted humanelements as the target absorber, for example, DNA, hemoglobin, and lipids [3,4]. Recently, the second generation of PA
tomography has adopted an exogenous high-absorption contrast agent to improve its contrast, spatial resolution, andfunctionality. This may extend the imaging depth from genes
to the whole human body [5,6]. In this letter, to accelerate thedevelopment of photoacoustic contrast agents, we report thedesign of a compact and cost-eﬀective test machine for thesensitivity evaluation of photoacoustic contrast agents. Thesystem adopts two laser diodes driven by a modulated
continuous source in the kilohertz region, and the generated
acoustic signal is detected by a small microphone attached onan acoustic resonator. The operation of the proposed setup isexamined using ink solutions and an existing contrast agent.
2. Design of photoacoustic evaluation system
2.1. Setup of photoacoustic evaluation system
Figure 1 shows the setup of the proposed evaluation
system for a contrast agent. Two laser diodes (LD) operating
at 660 and 785 nm are employed as excitation sources(660 nm LD, Mitsubishi ML101J27; 785 nm LD, Thorlabs
L785P090), and driven by a 100%-depth modulated voltage
using a function generator (Tektronix, AFG310) via an
appropriate resistor. The LDs are selected with a switch.
The average output optical power is set to approximately 20–
40 mW. The spot diameter is collimated to around 5.0 mm
using a collimator lens. We chose 660 and 785 nm as theworking wavelengths in this experiment, but these should beselected by consideration of the target materials. The twolaser paths are combined with a polarization beam splitter(PBS) and led to the sample liquid to generate a PA signal.
A one-dimensional PA cell is employed to enhance the PA
signal using the acoustic resonance of the cell. The bottom
end of the PA cell is in contact with the surface of the sample
liquid so that part of the PA signal generated in the sample
liquid can be led to the cell. Air is conﬁned in the PA cell. ThePA cell is made of copper and resonates in the ﬁxed-ﬁxed
mode since the top of the PA cell is sealed with a thin glasse-mail: xizeng@sonic.pi.titech.ac.jp

plate. A small microphone based on a micro–electro–mechanical systems (1:45  6:15  3:76 mm3 , Knowles Acoustics, SP0103NC3-3) is attached at the middle of the PA cell topick up the PA signal. The microphone covers the frequency
band from 100 Hz to 10 kHz. The PA signal is processed witha preampliﬁer (NF Corp., 3611) and a lock-in ampliﬁer (NFCorp., 5560).
2.2. Design of photoacoustic cell
The inner diameter and length of the PA cell are 5 and86 mm, respectively. The inner surface is polished tominimize the spurious PA signal due to the absorption on
the wall. The cell acts as a ﬁxed-ﬁxed acoustic resonator toamplify the PA signal. Resonance modes having a pressure
loop at the center can be used since the microphone is
attached at the center. The wavelength for the nth resonanceis written asn ¼2Lnðn ¼ 2; 4; 6 . . . . . .Þ;ð1Þusing the length of the PA cell L (¼ 86 mm). Then, the nthresonance frequency isf ¼nc2L

ðn ¼ 2; 4; 6 . . . . . .Þ:ð2Þ

Here, c is the sound speed.
2.3. Considerations for PA signal generation
PA phenomena in a liquid are categorized into six
diﬀerent cases according to the relationship between thesample length, optical absorption length, and thermal diﬀusion length [7,8].
In our experiment, the thermal diﬀusion length of the ink
solution is similar to that of water ( 0:0006 cm) owing to its
low concentration (<0:2%). We always keep the depth of thesample solution greater than the thermal diﬀusion length. A
laser ray penetrates through the sample. Thus, we have thefollowing relationship for the experiments reported in thisletter:u > l > us ;ð3Þ

where l is the depth of the sample, u is the absorption length,and us is the thermal diﬀusion length of the sample. The
intensity of the PA signal PAS and the other parameterssatisfy the following relationship according to Ref. [7]:I0    us
PAS /:
ð4Þ
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Acoust. Sci. & Tech. 39, 3 (2018)

Fig. 2 Resonant frequencies of PA cell measured forblack ink solution at 785 nm.

Fig. 1 Structure of photoacoustic evaluation system.
Table 1 Optical absorption coeﬃcients for water andinks in cm1 .
Wavelength(nm)

Water [9]Black ink

Dye ink

660
785

0.004
0.01

754.71203.41228.4590.47

Ratio(660/785)

0.40

3.7

2.5

Here, I0 is the incident monochromatic light ﬂux,  is theoptical absorption coeﬃcient of the sample, and ! is themodulation frequency. In our experiments, the PA signal isdetermined by the optical absorption coeﬃcient of the sample.
This is the key principle of the proposed PA system.
3. Characteristics of photoacoustic system
3.1. Absorption coeﬃcients of tested samples (inks)First, we tested solutions of a black ink, a dye ink,
and their mixture and a sample contrast agent to verify theproposed system. The absorption coeﬃcients of the inks
and the ratios are summarized in Table 1. The absorption
coeﬃcient of the black ink is larger than that of the dye ink
at both 660 and 785 nm wavelengths, while the absorption
coeﬃcients of water are much smaller than those of the inks.
The ratios of the absorption coeﬃcient at 660 nm to that at
785 nm were 3.7 and 2.5 for the black ink and dye ink,respectively.
3.2. Resonant characteristics of PA cell
Here, we tested the resonant frequency of the PA systemusing the black ink solution (45 mg/dl). The 785 nm LD acted
as a light source and its modulation frequency was varied
from 0.1 to 12.0 kHz with steps of 0.1 or 0.02 kHz. As shownin Fig. 2, the resonant frequencies were found approximately
at 4, 8, and 12 kHz, in agreement with Eq. (2). Considering

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Fig. 3 Duty ratio of the light source and PA output level.

the rated frequency range of the microphone (100 Hz to10 kHz), we chose the resonance at around 7.78 kHz as theworking frequency in this experiment. The working frequency
was adjusted to track the resonance in the range from 7.71 to
7.85 kHz since the temperature ranged from 17 to 26 C. Thequality factor of the resonance was estimated to be approximately 40. The response observed at very low frequencies isthought to be a spurious signal due to the electronic system.
3.3. Modulation duty ratio and PA signal
The duty ratio of the modulation for the LDs was alteredand the PA output was recorded. We carried out the testat 785 nm for water and a black ink solution (45 mg/dl) assamples. The results are shown in Fig. 3. The output signal
level increased with increasing duty ratio and showed amaximum at a duty ratio of 50%. This means that the sample
is continuously heated and expanding during the on-time of
the laser, and the output exhibits a maximum when theheating period is equal to the cooling period. In a future study,
we need to verify that the PA sensitivity evaluated by thismodulated method is identical to the sensitivity under singlepulse excitation.
4.

Evaluation of mixture ratio and concentration ofsamples
4.1. Concentration of black ink and PA signal
We gradually added black ink to a water sample (height,78 mm; volume, 2.35 dl) to increase the concentration of thesolution in a step of 10 mg/dl. The PA signals at both 660 andX. ZENG et al.: EVALUATION OF PHOTOACOUSTIC CONTRAST AGENT

Fig. 4 PA signals at 660 and 785 nm as functions ofconcentration of black ink solution.

Fig. 5 PA signals for solutions mixed with black anddye inks at 660 and 785 nm.

Table 2 Samples with black and dye inks mixed in0.5 dl water.
Sample group

012345Black ink (mg)
Dye ink (mg)002000150
50100100501500200785 nm wavelengths increased linearly with the concentrationin the range of 10–50 mg/dl as shown in Fig. 4. The average
optical power was adjusted to 20 mW. The signal level at
660 nm was always approximately three times that at 785 nm.
The results agreed well with the absorption coeﬃcients at
660 and 785 nm and the ratio between them shown in Table 1.
When the sample was water (0 mg/dl), the PA signal for
785 nm was higher than that for 660 nm because theabsorption coeﬃcient of water at 785 nm is higher than thatat 660 nm. However, the PA signal ratio for water at 660 and785 nm was not exactly the same as the absorption coeﬃcient
ratio of water because these low PA signals had alreadyreached the level of system noise, which was observed to bearound 80 mV.
4.2. Mixture of black ink and dye ink
In the second experiment, we prepared six groups ofsamples as listed in Table 2.
The ﬁrst sample was water and the other samples were ink
solutions composed of the black ink and dye ink. The results
are summarized in Fig. 5. From group 1 to 5, the quantity of
black ink decreased, while that of the dye ink increased. The
PA signals at 660 and 785 nm decreased from group 1 to 5
because the absorption coeﬃcient of the dye ink is smaller
than that of the black ink at both wavelengths. The PA signals
at 660 nm are always higher than those at 785 nm because theabsorption coeﬃcients of both inks at 660 nm are higher thanthose at 785 nm. Then, the ratio of the PA signal at 660 nm
to that at 785 nm was calculated and plotted in the ﬁgure.
The ratio of sample 0 (water) is less than one owing to its
absorption coeﬃcient ratio. The ratio decreased from group 1
to 5 in accordance with the mixture ratio since the absorption
coeﬃcient ratio of the dye ink at the two wavelengths wassmaller than that of the black ink. The mixture ratio of theblack ink to the dye ink could be estimated from the signal
ratio. Here, the average optical power for both wavelengths
was around 20 mW.

Fig. 6 PA signal level as a function of concentration ofICG solution.

4.3.

Test for ICG solution
Indocyanine green (ICG) is used for video angiography inhospitals owing to its ﬂuorescence characteristics. Here, wetested the photoacoustic capability of ICG solution using theproposed evaluation system. ICG was diluted by dimethyl
sulfoxide (DMSO), which is a common solvent and has little
absorption at these two wavelengths. A 4 ml sample wasprepared in a small glass dish of 20 mm diameter and 4 mm
depth. The PA signal was recorded as a function of theconcentration of the solution using the 660 and 785 nm LDs.
The absorption of ICG at 785 and 660 nm was 123,000 and21,200 Lcm1 mol1 respectively.
The PA signal increased almost proportionally to theconcentration at 785 nm as shown in Fig. 6. However, theresponses were not stable at higher concentrations. This might
be due to the poor stability of the sample. During themeasurement, the average optical power at 785 nm of
approximately 40 mW was injected into the sample, wherethermal and optical eﬀects excited by the optical power on
the sample must be considered. The PA signal at 660 nm
remained unchanged at lower levels around the noise level(around 80 mV) of the system because the absorption wasmuch smaller at this wavelength, and the injected opticalpower was 20 mW, which was half of that at 785 nm.
If ICG is mixed with another material, or injected into atissue with a strong-absorption chromophore, this PA systemwith two working wavelengths can determine the mixture
ratio of two materials as we demonstrated in the previous

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Acoust. Sci. & Tech. 39, 3 (2018)
experiment, or can determine the concentration of thechromophore from the concentration of ICG.
5.

Conclusions
A compact photoacoustic system using continuous excitation at a low modulation frequency was designed asevaluation equipment for PA contrast agents for medicalapplications. We conﬁrmed the successful operation of theprototype through measurements of diﬀerent ink solutions.
ICG solutions were also tested using the prototype. The
experiments reported in this letter were conducted under
uncontrolled room temperature and humidity. Moreover, thelaser power was not stabilized. It is necessary to introduce
regulated conditions both for the sample and the excitation forpractical use. In the near future, we need to compare theresults obtained by the proposed system and the quality of PA
images obtained by a practical PA imaging machine using
various contrast agents. We expect that the proposed simple
test setup will accelerate the development of new functional
contrast agents and be of help in pioneering a new generationof medical applications of PA imaging.

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