Determining The Optical Properties of Gelatine Gel

Determining the optical properties of a
gelatin-TiO2 phantom at 780 nm

H. Günhan Akarçay, Stefan Preisser, Martin Frenz, and Jaro Rička*
Institute of Applied Physics, University of Bern, Sidlerstrasse 5, 3012 Bern, Switzerland
*[email protected]
Abstract: Tissue phantoms play a central role in validating biomedical

imaging techniques. Here we employ a series of methods that aim to fully
determine the optical properties, i.e., the refractive index n, absorption
coefficient µa, transport mean free path  , and scattering coefficient µs of a
*
TiO2 in gelatin phantom intended for use in optoacoustic imaging. For the
determination of the key parameters µa and  , we employ a variant of time
*
of flight measurements, where fiber optodes are immersed into the phantom
to minimize the influence of boundaries. The robustness of the method was
verified with Monte Carlo simulations, where the experimentally obtained
values served as input parameters for the simulations. The excellent
agreement between simulations and experiments confirmed the reliability of
the results. The parameters determined at 780 nm are = n 1.359(±0.002) ,
=µ s′ 1=
/ * 0.22(±0.02) mm -1 , µ a = 0.0053(+0.0006-0.0003) mm -1 , and
=µ s 2.86( ± 0.04) mm -1 . The asymmetry parameter g obtained from the
parameters  and µ s′ is 0.93, which indicates that the scattering entities
*
are not bare TiO2 particles but large sparse clusters. The interaction between
the scattering particles and the gelatin matrix should be taken into account
when developing such phantoms.
© 2012 Optical Society of America
OCIS codes: (170.3660) Light propagation in tissues; (170.6935) Tissue characterization;
(290.1990) Diffusion; (290.4210) Multiple scattering.
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1. Introduction
The development of diagnostic imaging techniques commonly entails the use of “phantoms”,
which try to mimic human tissue as closely as possible. They are conceived in such a way that
their optical properties are similar to those of tissues and, therefore, constitute a precious tool
which provides a well-defined environment to study light propagation. Quoting a recent
comprehensive review by Pogue and Patterson [1], these phantoms “are used for a number of
purposes, including: (i) initially testing system designs, (ii) optimizing signal to noise in
existing systems, (iii) performing routine quality control, (iv) comparing performance
between systems.”
Our group is conducting research in optoacoustic (OA) imaging [2] where phantoms are
widely used based on hydrogels (such as gelatin, agar, polyvinylalcohol) or PVC plastisol and
other resins (see [1,3–5] and references therein). We opted for gelatin phantoms because they
are relatively easy to produce and have the advantage of being adjustable to various purposes:
(i) the phantom's optical properties can be customized by incorporating scattering agents (like
TiO2 or intralipid) or absorbing elements (like India ink, or any dye of interest), and (ii) the
phantom's mechanical/elastic properties can be modified by varying the fraction of gelatin.
Elastic properties especially play a valuable role: indeed, the combination of OA imaging with
ultrasound elastography holds promise for reduced clutter and increased imaging depth in the
framework of clinical imaging [6,7].
Our gelatin phantoms are produced in a two-step approach. First, a phantom precursor
stock is prepared by dissolving gelatin in water and by incorporating titanium dioxide (TiO2)
as the scattering component. During the second step, different phantoms can be developed
from this precursor stock by adding various amounts of supplementary scattering agents,
absorbing structures, and acoustic scatterers (such as flour) to obtain more realistic optical and
acoustic properties for combined OA and ultrasound imaging.
Accurate knowledge of the optical properties of the phantom precursor stock is essential
within this process. Determining the optical properties of such phantoms or tissues is not an
obvious task and has been subject to intensive research over the past decades (see e.g. the
review by Kim and Wilson [8]). In this paper, we employ a series of methods by which we
were able to fully characterize the optical properties of the precursor stock (which is simply
referred to as “phantom” or “sample” throughout this manuscript) at a wavelength of 780 nm.
This wavelength is particularly appropriate since it lies within the diagnostic window where,
owing to low optical absorption, OA imaging can achieve several centimeters of imaging
depth [9].
Fig. 1. Diagram summarizing the different stages of the work presented in this paper: the
optical properties (refractive index, transport mean free path, absorption coefficient, scattering
coefficient, and scattering law) of the phantom were experimentally determined by the
indicated set of experiments. Monte Carlo simulations were performed ex-post to validate the
experimental results.
The way this paper is organized is outlined in the diagram of Fig. 1. After describing how
the gelatin phantom was produced, we describe how the optical properties of the phantom
were determined at 780 nm, as follows:
a) its refractive index n was determined with an Abbe refractometer;
b) its transport mean free path * and absorption length  a = 1/ µ a (where µa is the
absorption coefficient) were measured by performing time of flight (TOF)
measurements within the phantom;
c) its scattering coefficient µs was assessed from extinction measurements with an
absorption spectrometer;
d) and its scattering law (which models the way the phantom’s scatterers spatially
redistribute the light) together with its corresponding asymmetry (or anisotropy)
parameter g were evaluated from goniometric measurements.
In the last part we present Monte Carlo (MC) simulations performed to validate the
experimental results: we simulated the TOF experiments by providing the experimentally
determined optical properties of the phantom as inputs to the MC program.
2. Gelatin phantom preparation
2.1. Ingredients and preparation of the phantom
The phantom precursor mixture consists of 16.7% weight fraction of gelatin dissolved in
water and 1 gram per liter (g L−1) of TiO2 for optical scattering. The TiO2 concentration was
chosen according to the data in [4], which suggest a reduced scattering coefficient of 0.4 mm−1
at 1 g L−1. This is below the range 0.5-1.3 mm−1 typically found in human breast tissue
[10,11], but we anticipate the need for further additives, including e.g., flour for acoustic
contrast, which will modify the scattering properties.
The TiO2 particles were obtained from Millenium Chemicals (type Tiona RL11A). They
consist of 99% rutile, with a refractive index of 2.4884 at 780 nm [12]. Their size distribution
lies between 180 nm and 400 nm diameter, with a clear peak at 250 nm (weight percentage,
determined by disc centrifugation).
The phantom in its precursor form is prepared as follows:
• With the help of an agitator, 1 kg of gelatin is mixed with 5 L of a stock solution
prepared with deionized water and composed of 1 g L−1 TiO2 and 1 g L−1
methylparabene (which serves as preservative), so that the final phantom contains
16.7% weight percentage of gelatin. (For the dilution series used in Sections 4 and 5,
the aqueous TiO2 dispersions are pre-diluted to the required TiO2 concentrations.)
• The mixture is heated to 60°C, so that the gelatin gets dissolved and the mixture
becomes homogeneous.
• After heating the mixture up to 60°C, it is left to cool down to 50°C, with the agitator
running throughout the whole heating and cooling processes, in order to maintain the
homogeneity of the mixture.
• Finally, the resulting bulk solution is poured into a glass aquarium (of size
180 mm × 240 mm × 210 mm : see Fig. 2) where it cools down to room temperature
and becomes solid. As explained in Section 3, the phantom is prepared in such a
large container in order to avoid boundary condition problems during the TOF
measurements
2.2. Refractive index
For the evaluation of TOF measurements (Section 3) and for the MC simulations (Section 6),
it is necessary to know the refractive index n of the phantom at the wavelength
of λ = 780 nm . After having prepared a phantom sample, we measured at the room
temperature of 22°C both its refractive index nD ≈ 1.363 at the sodium d-line wavelength
λD = 589.29 nm and its corresponding Abbe number υ= 1 / (nF − nC )= 56.72 , where nF and
nC are the refractive indices at the wavelengths λF = 486.13 nm and λC = 656.27 nm . These
two values were then used to determine the two parameters A and B of the two-term form of
the Cauchy extrapolation formula n(λ )= A + B / λ 2 . This law gives the refractive index of the
phantom at 780 nm:= n 1.359 ± 0.002 . The measurements were done with an Abbe
refractometer (Type 1T by ATAGO). Since the refractrometer is calibrated with water, we use
the error estimate of the IAPWS formulation for pure water in the wavelength range 700-1100
nm [13].
3. Determination of the transport mean free path and absorption length
3.1. Principle of the measurements
Our technique is a variant of position and time resolved diffuse reflectometry [14], but we
place photon injection and detection deep inside the scattering medium, in order to minimize
the influence of the boundaries [15–17]. The principle is illustrated in Fig. 2. Photons
generated by a pulsed picosecond diode laser (LDH-P-785, PicoQuant) are delivered into the
phantom through a multimode optical fiber (50 mm core diameter) and injected at a point S
well inside the scattering medium. A second fiber collects the scattered light at a point D at a
distance r from S and guides it to a single photon detector (id100-50, ID Quantique). The time
of flight (TOF) of the individual photons from S to D is measured by a fast timer (SPC 530
PicoQuant) triggered by the laser pulse. The resulting TOF distribution is characteristic of the
optical properties of the material through which the light propagates. Thus, by fitting an
appropriate analytical model to the recorded distribution curve, the optical properties of the
phantom can be extracted.
Fig. 2. Schematic representation of the principle of the time of flight (TOF) experiments: the
light source – a picosecond laser – injects the light into the phantom at a point S; the individual
photons are then subject to multiple scattering in the phantom (random photon paths are
represented in red) until they exit the phantom or get absorbed in it. The photons that reach the
detection point D (placed at a distance r from S) are detected and their individual TOF from S
to D recorded in a histogram. This experiment is reproduced for different source-detector
separations r and the obtained TOF distributions are then fitted to the diffusion model.
3.2. Diffusion model

We employ the photon diffusion model [18], which is an approximation to describe the light
transport in strongly scattering media (i.e., where µ s >> µ a ) and over large distances such
that r >> * . According to this approximation, the probability P (r, t )dr 3 dt to detect a photon
in a volume element dr 3 and time interval dt obeys the diffusion equation:
∂P (r, t )
−α p P (r, t ) + S (r, t )
− D p ∇ 2 P (r, t ) = (1)
∂t
where D p is the photon diffusion coefficient and α p is the absorption rate. D p and α p are
related to the phantom’s transport mean free path * and absorption length  a respectively:
1 * cl
=Dp =cl  and α p . (2)
3 a
Here cl is the light speed in the medium. (In the biomedical photonics community one often
uses the reduced scattering coefficient µ s′ = 1/ * and µ a = 1/  a . Note that we assume
µ s >> µa .) The Green’s solution of Eq. (1) is
1  −r 2 
P=(r, t ) ⋅ exp   ⋅ exp(−α p t ) . (3)
(4π D p t )3/ 2  4Dp t 
This simple solution applies in infinite space and for a δ-source in space and time:
S (r, t ) = δ (r )δ (t ) . To approximate the former condition, we employ a large sample container,
so that photons are injected and detected far from boundaries. To regard S(r) as a point
source, we require that ds  * , where ds is the characteristic size of the source. This
condition is easily met by using an optical fiber as an illuminator.
The time dependence of the measured distribution M(t) results from the convolution of
P(t) with the instrument response function IRF(t) that includes the finite width and shape of
the laser pulse and the timing jitter of the detection: = M IRF ⊗ P . To determine the least
square estimates of the parameters Dp and αp, we apply the standard iterative reconvolution
technique, using the Levenberg – Marquardt algorithm provided by Matlab. A third parameter
is needed to account for the transition from the ballistic photon transport to diffusion that
occurs within approximately a distance of 2 * from the injection point. We include in the
fitting procedure a (manually) variable shift τd of the measured TOF-distribution with respect
to the measured IRF. This shift does not play a significant role if r >> * . Since * is not
known a priori, we must carry out a series of measurements at varying source-detector
separations. These estimated values should converge to the “true” values as r increases, so
that most of the photon paths are long enough to assure the validity of the diffusion
approximation.
3.3. Setup and measurement procedure
The phantom is contained in a soda-lime glass aquarium ( 180 mm × 240 mm × 210 mm ). The
setup was designed to perform measurements at different source-detector separations without
deforming either the phantom or the fibers (see Fig. 3). The multimode fibers (core diameter
of 50 mm), both on the laser and the detector side, are guided through hypodermic needles
(diameter 0.8 mm) in order to reach the inner parts of the gelatin phantom with minimal
damage and better accuracy. Up to eight such optodes can be positioned in the orifices of a
holder-plate and fixed perpendicularly to the phantom’s top surface. The heads of the needles
are placed on the holder so that their tips are deep inside the phantom. This allows both the
injection and the collection of the light far from the phantom-air boundary (typically at a
depth of 40 mm beneath the top surface). To avoid distortions caused by the insertion of the
needles, the phantom was poured in its liquid state into the aquarium where the holder and
fibers had been positioned beforehand; the phantom then solidified with the needles already in
place. The accuracy of the positioning of the fiber tips was inspected by taking photographs
prior and after the solidification of the gelatin: we found a maximum deviation of 0.6 mm
from the intended separation.
Each pair of fiber/orifice on the holder plate corresponds to a different source-detector
separation r. Thus, measurements at different source-detector separations are carried out by
successively selecting different pairs of fibers and connecting each fiber to the laser and the
single photon counting module. Prior to each measurement the IRF corresponding to the
chosen pair of optodes was determined by measuring the time-resolved scattering from a sheet
of white paper immersed in water. With the present laser and photon detector the width of the
IRF is small when compared with the width of the TOF distribution M(t). Therefore, the
precise shape of the IRF does not play a crucial role. What matters, however, is the timing,
i.e., the shift between IRF(t) and M(t). Strictly speaking, for a precise determination of the
IRF, one should place the fiber tips close together so that the photon path length is shorter
than 1* . However, this exact positioning is not easy to achieve (for example, we must insert
filters to adjust the photon-count rate), and, therefore, in the data evaluation we shall rely on
the adjustable parameter τ d to compensate the resulting inaccuracy of timing. An example of
the best fit of the measured data by the diffusion model is shown in Fig. 4.
Fig. 3. Detail of the setup used for the TOF measurements. (a) The gelatin phantom (5 L) is
contained in a soda-lime glass aquarium. (b) and (c) In the holder-plate, the optodes can be
placed at eight positions. Each pair of optode/orifice on the holder-plate corresponds to a
different source-detector separation r. The tips of the needles are inside the phantom, at a depth
of 40 mm beneath the phantom/air interface.
Fig. 4. Example of the fit of the measured data (thin solid line in black) with the diffusion
model (thicker line in red). The corresponding IRF is shown in green dots. For comparison, we
also include the fit with the corresponding MC simulations (see Section 6) with the same
parameters  and  a (thicker line in blue).
*
3.4. Results and discussion

In Fig. 5 we compile the results of five series of measurements on five different phantoms.
First of all we observe that * is rather large, larger than the transport mean free path in
human breast (typically around 1 mm [10,11]). Correspondingly large is the influence of
inaccuracies and limitations of the diffusion approximation, which results in the data scatter at
small distances r. From an analysis of the influence of primary experimental errors on the
least square estimates of the parameters * and  a we found that the overall variance is
composed of three contributions:
i) Accuracy of the fiber positioning, whose influence on * depends on the optode
distance r. For r = 20 mm , an error in r of 1 mm results in 0.4 mm error in the
estimation of * , at r = 50 mm , the resulting error is only about 0.15 mm. The
impact of the positioning error on  a is negligible.
ii) Accuracy in the determination of the parameter τ d , i.e., the shift between the IRF(t)
and M(t). With an optode distance r = 20 mm , an error in τ d of 50 ps will lead to
0.5 mm misestimation of * , at r = 50 mm the resulting error in * decreases to
about 0.3 mm. In addition, the parameter τ d accounts for about 50% of the variation
of  a .
iii) The remaining variation of  a and * (see e.g. the outlier, phantom 3 at 43 mm
distance) is probably due to imperfect reproducibility of the fabrication and, in
particular, to the inhomogeneity of the phantom. One likely cause of the
inhomogeneity is the sedimentation of the TiO2 particles during the
cooling/solidification process.
Fig. 5. The values of the transport mean free path (top) and absorption length (bottom) obtained
from the TOF measurements with five different phantoms. The vertical dashed line is a “guide
to the eye” indicating roughly the validity limit of the diffusion approximation.
Nevertheless, the estimates of * of the five phantoms converge with a reasonable consistency
to the values =* 4.5 ± 0.5 mm and =  a 190 ± 15 mm , as r increases beyond approximately
six times  .
*
The transport mean free path of almost 5 mm (reduced scattering coefficient µ s′ of only
0.2 mm−1) is substantially larger than the typical values reported for elastomer based
phantoms with the same TiO2 concentrations (DMSO 0.9 mm [19], PVCP 2.5 mm [4],
Polyurethane 1.25 mm [20]). On the other hand, much larger values for * have been reported
for a comparable gelatin based system: Pogue et al. [21] measured µ s′ (c) of TiO2 in 7%
gelatin (at λ = 690 nm ) and found a linear concentration dependence µ s′ (c) ≈ 0.14 ⋅ c ( µ s′ in
mm−1, c in g L−1) up to c = 40 g L−1. Their data suggest that * is as large as 7 mm for 1 g L−1
TiO2 (unfortunately, the particle size is not specified).
4. Scattering coefficient and asymmetry
4.1. Extinction measurements
The extinction coefficient µe of the phantom was determined at 780 nm using a Perkin Elmer
LAMBDATM 750 absorption spectrometer. With the absorption coefficient µ a = 1/  a as
small as 0.005 mm−1, we expect that most of the extinction is due to scattering, i.e., µe = µ s .
If the concentration of the TiO2 solution is low enough that multiple scattering can be
neglected, then the transmission follows Beer-Lambert’s = law I I 0 exp[−d µ s (c)] , where d is
the thickness of the sample, I 0 is the intensity of the illuminating light and I the intensity of
the transmitted light. Furthermore, µ s (c) depends on the concentration c of the TiO2 particles.
In the simplest case, i.e., when concentration dependent clustering of titania particles can be
neglected, µ s is proportional to the TiO2 concentration c in the sample:
µ s (c) = c σ s _ TiO 2 (4)
where σ s _ TiO 2 is the scattering cross-section. Thus, the absorbance A = − log10 ( I / I 0 ) is
expected to be linear in d and c: A = log10 (e) σ s c d . The experimental data shown in Fig. 6
show that A is indeed linear in both, the sample thickness d and concentration c, which
indicates that we indeed measured in the single scattering regime and that the cross-section of
the individual scattering entities does not change with the concentration. Moreover, the linear
regime characteristic of the Beer-Lambert’s law extends up to the concentration c = 1 g L−1,
which we used to prepare the phantom.
The scattering coefficient at a TiO2 concentration of 1 g L−1 as obtained from the linear fit
to the data in Fig. 6 is µ= s (c
−1
1 g L= ) 2.86 ± 0.04 mm -1 . The error estimate represents the
standard statistical error of the least square parameter estimation.
Fig. 6. The absorbance A of TiO2 solutions (containing 16.7% weight percentage of gelatin) as
the function of concentration c (left: sample thickness d = 0.1mm; right: d = 1mm).
4.2. Asymmetry parameter g
Knowing the scattering coefficient μs, we can determine the phantom’s asymmetry parameter
g = cos(θ ) , i.e., the average of the cosine of the scattering angle θ , which is related to the
transport mean free path and the scattering coefficient according to
1 1
=
=
* (5)
µ s′ (1 − g ) µ s
Using * = 4.5 ± 0.5 mm, and µ s = 2.86 ± 0.04 mm−1, we obtain= g 0.93 ± 0.01 . This
large asymmetry indicates substantial forward scattering, which is much more pronounced
than one would expect from Mie scattering on TiO2 particles. The largest g one can get with
isotropic spherical particles of the given refractive index ratio would be about 0.7 (regardless
of the size of the particles!). Moreover, finite element calculations of Thiele and French [22]
indicate that the effect of non-spherical shape and anisotropy is not sufficiently large to
explain the large asymmetry. Apparently, the scattering entities are not bare isolated TiO2
particles. We speculate that the effective scatterers are some large (though sparse) TiO2-
gelatin clusters formed by micro-phase separation during the solidification of the system.
5. Assessing the scattering law
The ultimate characterization of a phantom tissue would consist of the complete determination
of the scattering law p (θ , φ ) (also called “phase function”) characterizing the angular
distribution of scattered photons in a single scattering event. With samples exhibiting strongly
asymmetric, nearly forward, scattering, this is a rather difficult task, which requires
specialized instrumentation. Here we report preliminary results obtained with a commercial
goniometer originally intended for dynamic light scattering measurement (ALV GmbH
Langen). The available hardware does not allow a precise determination of p (θ ) at small
angles, but is sufficient to check the consistency of the results obtained so far.
5.1. Goniometric measurements
The geometry of the scattering experiment is sketched in Fig. 7. The light source was a 780
nm CW diode laser (Laser Components). The scattered photons were collected with a
multimode fiber receiver focused at the center of the sample and counted with a single photon
detector. The sample is immersed in a toluene filled cylindrical vat, to minimize geometrical
Fig. 7. Top view of the light scattering experiment. The light emitted by the laser is vertically
polarized. We measured the light scattered by TiO2 suspensions (containing 16.7% weight
percentage of gelatin) of different concentrations at various angles 40°≤θ≤140°.
errors resulting from refractive index mismatch. A polarizer-analyzer pair selects the SS-
component (perpendicular to the scattering plane) of the phase function.
An accurate determination of the single scattering law of the strongly scattering sample
would require an extrapolation to a sample cell whose dimensions are smaller than 1 / µ s , i.e.,
smaller than 0.4 mm. This is not practicable with the given setup and even less with a
heterogeneous sample. However, the observed linearity of µ s (c) and µ s′ (c) (see [21])
indicates that the structure of the scattering entities does not depend on the TiO2
concentration. This suggests a simpler alternative: We carried out measurements on TiO2
dispersions (each containing 16.7% gelatin) with decreasing concentrations, and from this
series we extrapolated the trend of the scattering law. Examples of the extrapolation procedure
are indicated in the inset in Fig. 8.
For each TiO2-gelatin dispersion, the scattered intensity I meas (θ ) was measured at
different angles 40o ≤ θ ≤ 140° . (The apparatus is not optimized for forward scattering and
therefore we restrict the measurements to scattering angles larger than 40°). The measured
signal I meas (θ ) is normalized, so that I n (θ ) is obtained as follows:
I meas (θ ) − I blank (θ )
I n (θ ) = (6)
I tol (θ )
Here I blank (θ ) is the blank signal, measured with water in place of the TiO2-gelatin sample
and I tol (θ ) is the reference signal measured with toluene. Since I (θ ) ∝ µ s p (θ ) , the
normalized signal can be expressed as:
µ s p(θ )
I n (θ ) = (7)
µ s _ tol ptol (θ )
µ s _ tol and ptol (θ ) are the scattering coefficient and the scattering law of the toluene,
respectively and µ s (c) is the concentration-dependent scattering coefficient of the phantom as
previously determined. Toluene scatters as an almost perfectly isotropic dipole scatterer
(Rayleigh scattering), thus: ptol (θ ) = 3 / 8π . (The theoretical background of polarized Rayleigh
scattering is discussed for example in [23].) Thus, the scattering law of the TiO2 suspension
can be determined according to
3 µ s _ tol
p (θ c) = I n (θ ) (8)
8π µ s (c)
Moreover, µ s _ tol is identical to the so-called Rayleigh ratio Rtol of the toluene. From the
data provided by Moreels et al. [24], we determine the Rayleigh extrapolation formula
λ ) 1.65 × 108 / λ 4 (Rtol in m−1 and λ in nm), which leads to µ s=
Rtol (= _ tol 4.46 × 10−4 m -1 . In
Fig. 8 the extrapolated values are indicated with symbols “+”. Note, that since µ s _ tol , ptol (θ )
and µ s are known, an adequate model should match not only the shape but also the
magnitude of the experimental curve without adjusting a normalization pre-factor.
5.2. Modeling of the scattering law
As the first guess, an obvious model would be the Mie scattering law corresponding to nearly
spherical TiO2 particles with a certain size distribution. However, as already mentioned,
disregarding the size of the particles, the largest value of g that can be obtained with TiO2
dispersed in the gelatin gel is 0.7. This is much smaller than the previously determined g =
0.93. Thus, we need another scattering law to model the proposed clustering of the TiO2 in the
gelatin. To account for the interference between the individual scatterers forming a cluster, we
resort to the Rayleigh-Debye approximation. (Which is also suited to describe light scattering
in tissue in a more realistic fashion than Mie scattering. See Rička et al. [23].) In the
Rayleigh-Debye approximation one combines polarized scattering from elementary Rayleigh
scatterers with a structure factor that characterizes their mutual arrangement. We employ the
polarized version of the generalized Henyey-Greenstein scattering law (pvgHG) (discussed in
more details in Refs. [23,25]), which assumes the following “fractal” structure factor F:
1
F (q | L,ν ) ∝ where ν > −1 (9)
[1 + ( L | q |) 2 ]ν + 3/ 2
Here q is the scattering vector,= |q|2 2k 2 [1 − cos(θ )] , where k is the wavenumber in the
medium and θ the scattering angle. The pvgHG structure factor has two modifiable
parameters: the correlation length L characterizes the extent of correlation between the
elementary scatterers, i.e., roughly the size of the proposed clusters. The power law exponent
ν is related to the shape of the correlation, i.e., roughly the shape of the clusters. The case
ν = 0 corresponds to the well-known Henyey-Greenstein phase function widely used in the
field of tissue scattering. (Note, however, that we also include polarization. For more on these
topics see Section 7.3 in [23].) The correlation length L is not yet known, but we already have
an estimate of the asymmetry parameter g. Therefore, for the convenience of the data analysis,
we numerically convert the parameters { L,ν } into { g ,ν } .
Fig. 8. Estimating the scattering law of the TiO2 scatterers immersed in gelatin: the pvgHG
scattering law model is fitted to the extrapolated data (represented by “+” symbols in the main
figure) corresponding to infinitesimal concentrations of TiO2, i.e., where the single scattering
regime applies. The fitting is achieved by modifying the set of parameters {g, ν}. The
extrapolation procedure which gives the values corresponding to infinitesimal concentrations is
indicated in the inset figure.
Thus, the pvgHG model is fitted to the experimental data by manually modifying the set of
parameters { g ,ν } . (Recall that { g ,ν } are the only adjustable parameters; there is no adjustable
pre-factor.) The examples in Fig. 8 indicate the sensitivity to the parameter g. The model with
g = 0.90 and ν = 0 (curve 1) fails to give an accurate description of the data but with g = 0.93
(curve 2) is more satisfactory. A further improvement is achieved by varying the parameter ν.
A reasonably good fit is obtained with { g = 0.93,ν = −0.3} (curve 3).
From g = 0.93 we estimate a correlation length L in the order of 10 µm. The exact value
depends on the parameter ν, but the data quality does not allow its unambiguous
determination. Nevertheless, the scattering law is consistent with the previous results from
Sections 3 and 4.
6. MC simulations
In the last stage of the investigation, MC simulations were performed to assess the robustness
of the experimental results. We employ a simulation tool that we recently developed [25] to
study polarized light propagation in complex geometries. While polarization is not important
in the diffusion limit, we shall take advantage of the following features of our software:
i) A user interface allows creation of complex shapes in the simulation space through
which the ray tracing algorithm follows the paths of photons across the interfaces and through
the different regions. ii) The user can choose the optical properties for different regions of the
sample, including the interface properties (e.g. Fresnel reflections) and scattering laws.
(Currently Mie and pvgHG are implemented.) These features allow transposing the TOF
experiment into a realistic simulation (compare Fig. 9 and Fig. 2). The optical properties of
the phantom and its container that were given as inputs to the simulations are listed in Table 1.
To simplify the simulation design and to speed up the computation we permit ourselves
certain simplifications of photon launching and detection, which should not affect the results
in the diffusion limit. Specifically, we neglect the presence of the needles, because they are
made of non-absorbing material and thus create only a minor disturbance.
Photons are simply launched downwards at a position corresponding to the tip of the
source fiber, 40mm beneath the phantom’s top surface. The individual photons are launched
with randomly chosen initial polarization (the so-called unpolarized light) to mimic the
depolarization of the laser light occurring in a multimode fiber.
Fig. 9. Screenshot from the MC simulation: the simulation tool was used to reproduce the TOF
measurements presented in Section 3 (depicted in Fig. 2). The colored disc visualizes the
number of photons detected by the individual detector rings.
The detection is simulated as a concentric array of detectors (r resolution of 1mm), which
register incoming photons without absorbing them (see Fig. 9). We used two versions of this
detection scheme. In the first version, a photon is registered when it crosses the horizontal
plane of the receiving fiber tip within a numerical aperture NA = 0.87. The fibers used during
the measurements have NA = 0.2, but increasing the NA reduces the simulation time. The
second detection scheme registers a photon when a scattering event occurs within a narrow
slab (thickness 1 mm) adjacent to the detector plane, again into the aperture of 0.87. (To
compare the resulting TOF distributions for different scattering laws, they must be normalized
by dividing with μs.) This second detection scheme provides approximately a six fold
improvement of counting statistics but is only appropriate for distances within the validity of
the diffusion approximation.
All the simulations presented in this paper have been performed on a Linux PC equipped
with an Intel Pentium D computer processor with a clock speed of 3.40 GHz and a cache size
of 2048 KB. The path generation routine written in C + + has been compiled with a GNU
compiler (gcc version 4.3.2).
Table 1. The optical properties (at λ = 780 nm) assigned to the three different materials
used in the experiment: the quantities listed here correspond to the experimental results
and were provided as input parameters to the MC simulation program
Parameters Phantom Glass Aquarium Air

Refractive index 1.359 1.52 1.0
Transport mean free path *input 4.5 mm ∞ ∞
Absorption length  a _ input 190 mm ∞ (no absorption) ∞ (no absorption)
−1
Scattering coefficient 2.86 mm ∞ (no scattering) ∞ (no scattering)
6.1. Testing the diffusion limit, influence of scattering law

In a first series of simulations we tested whether the setup indeed fulfills the conditions for the
applicability of the diffusion approximation. The results for two optode distances r are shown
in Fig. 10. The transport mean free path and absorption length are set to the previously
determined values *input = 4.5 mm ,  a _ input = 190 mm , but we vary the scattering law (see inset
in the bottom of Fig. 10) and its parameters. Obviously, at r = 5 mm the diffusion
approximation fails to describe the simulated data (which is not surprising, see e.g [26].), but
for 36 mm distance (8 * ) we are already well within the diffusion regime. Moreover, at
r = 36 mm there is no way to distinguish the scattering law used in the simulation, since *
and  a are the only parameters that matter in the diffusion limit. However, the influence of
the scattering law becomes apparent at an optode separation of about 1 * , despite the fact that
all simulations are done with the same * and  a . Apparently, the asymmetry g is important
on the length scale * and on the time scale that corresponds to a mean photon path length of
1 * . The asymmetry g as well as the form of the scattering law affect angularly resolved
reflectance experiments [27,28].
6.2. Analyzing the simulated data
In Fig. 4 the outcome of the simulation (pvgHG law { g = 0.93,ν = −0.3 }) is compared with
the measured data obtained at the optode separation r = 36 mm . The simulation is convoluted
with the IRF of the experiment, i.e., we regard the simulation as a model function M(t), an
alternative to the diffusion model. Both models match the measurement within the statistical
error. However, there is a slight mismatch between the diffusion model and the simulation
model with the same parameters. This could indicate that at r = 36 mm we are not yet within
the range of the validity of the diffusion approximation.
Fig. 10. Examples of the MC simulations where we varied the scattering law and its
parameters. The curves in the inset of the bottom figure are polarization averaged. The data
were generated in an overnight simulation including 60 million photons for each scattering law
(total number of scattering events: 47 × 109). The simulated curves show the raw, non-
normalized and unsmoothed data with a time-resolution of 2 ps. At small source-detector
separations (top), the limitation of the diffusion model (red line) is clearly visible but the
differences between the scattering laws are hardly discernable in the semi-log plot. These
differences are significant at short times, as shown in the linear plot in the inset of the top
figure. (The small bump at 25 ps is probably a commensurability artifact due to the finite
resolution of the ring detector.) At large source-detector separations (bottom), the applicability
of the diffusion model becomes apparent. At both distances, the diffusion model is shifted by τd
≈50ps.
To further elucidate this issue, we analyzed the simulation data by fitting the diffusion
model. Note that thereby we regard the simulation data as equivalent to a measurement, the
only difference from a real measurement is that we have now a perfect estimate of the IRF,
namely a δ-peak positioned at t = 0. The fit parameters are the transport mean free path * ,
absorption length  a and the shift τ d that accounts for the transition from ballistic to
diffusive regime of photon propagation. Examples of the results for four optode distances are
summarized in Table 2. The estimates of the parameter uncertainty are derived from visual
inspection of the quality of the fit. (Statistical analysis in terms of χ2 would not be appropriate,
since we expect a systematic discrepancy between the diffusion model and the data for short
photon paths.)
The optimal shift τ d = 50 ps , corresponding to 11 mm of the photon path, is consistently
found for all optode separations. Thus, the ballistic regime of photon propagation extends to
about 2 * . Note, that the uncertainty of the parameter τ d increases with the optode distance.
This shows that the shift ceases to be important as the spacing enters the range of validity of
the diffusion approximation. The parameter * converges within the given uncertainty to the
input value *input = 4.5 mm . However, the parameter  a converges to a value which is
significantly smaller than the input value  a _ input = 190 mm . This indicates the increasing
influence of the boundaries on this parameter. Apparently, the optode depth of 40 mm below
the air interface is too small, causing a cut-off of long photon paths. Thus, the  a measured
with the real phantom is slightly overestimated (which we take into account in the final error
estimate in Section 7, Conclusions). However, the resulting error of about 10 mm remains
well within the experimental error estimate.
Table 2. Parameters obtained by fitting the diffusion model to the simulated data
= =
with  input 4.5 mm,  a _ input 190 mm
*
Transport mean Absorption length

Optode distance
r [mm] Shift τ d [ps] path * [mm]  a [mm]
24 50 ± 5 5.5 ± 0.5 214 ± 16
36 50 ± 6 4.8 ± 0.3 186 ± 8
48 50 ± 14 4.6 ± 0.2 179 ± 5
55 50 ± 20 4.6 ± 0.2 178 ± 3
7. Conclusions
The optical parameters of our phantom tissue precursor consisting of 1 g L−1 TiO2 in 16.7%
gelatin are summarized as follows:
Refractive index:=
n780 1.359(±0.002) ;
* 4.5(±0.5) mm [ µe′ ≈ µ s′= (0.22 ± 0.02) mm −1 ];

Transport mean free path: =
Absorption length:  a = 190(+10 − 20) mm [ µ a = 0.0053(+0.0006 − 0.0003) mm -1 ];
Extinction coefficient: µe ≈ µ s = 2.86(±0.04) mm -1 .
The transport mean free path * of almost 5 mm (reduced scattering coefficient µ s′ of only 0.2
mm−1) is substantially larger than initially expected for the given concentration of TiO2
particles. This large * reflects the large asymmetry of underlying scattering law. From the
measured µ s′ and µ s we obtain g = 0.93( ± 0.01), which is much larger than one would expect
for Mie scattering from bare TiO2 particles (g = 0.5-0.7). Indeed, our preliminary
measurements with a simple goniometer indicate that the angular dependence of scattering is
not compatible with the TiO2 particles, but can be reasonably modeled with a generalized
Henyey-Greenstein structure factor (Eq. (9) with g = 0.93 and shape parameter ν = −0.3 . We
speculate that the scattering is due to sparse microstructures in the order of 10 μm in size.
These findings must be taken into account in the next steps of the phantom development.
When designing a tissue phantom, one should not blindly expect the optical properties of the
product to be the sum of the optical properties of its components. The optical properties
depend on the microstructure of the system, as it results from interactions between the
constituents, and whose formation depends on the physico-chemical parameters such as pH
and ionic strength. Certainly, the microstructure of the gelatin-TiO2 phantoms would deserve a
thorough investigation. (This, however, would require a dedicated small angle light scattering
setup.)
The parameters * and  a (which are the relevant parameters in many practical situations)
were determined using a novel version of the fiber-optic TOF technique in which we immerse
the injection and detection optodes deep inside the scattering medium. This geometry
minimizes the influence of the interfaces on the TOF distribution and allows the analysis of
the data in terms of the simple free space diffusion approximation.
The robustness of the approach was tested using our new simulation tool, which allows not
only realistic modeling of the experimental geometry but also provides a simple way to vary
the scattering law and its parameters. In the diffusion regime, the parameters obtained from
simulated and experimentally determined data agree well within their error estimates. The
simple diffusion approximation can be reliably applied down to optode separations of less
than 6 * , if one corrects for a small time shift τ d ≈ 2* / cl (in our case about 50 ps) that
accounts for the transition from ballistic to diffusive regime of light propagation.
Moreover, our simulations confirm that the above four parameters, namely n, * ,  a and
μe, represent a necessary and sufficient set of optical parameters of a realistic tissue phantom.
Usually n, * ,  a are the most relevant parameters. The fourth parameter μe (or rather the
asymmetry parameter g that corresponds to a certain μe when * is fixed) plays a role only at
length and time scales that involve a small number of scattering events. (This may be the case,
for example, with multiphoton imaging of the skin.) In any case, the exact shape of the
scattering law seems to play a negligible role in most practical situations (although there may
be exceptions [27]). This certainly simplifies the design and characterization of the phantom
tissue.
These insights combined with the experimental and computational tools described here
create a powerful platform on which to build realistic tissue phantoms. Such optically,
acoustically, and geometrically realistic phantoms will serve as essential tools in the
development of important new biomedical imaging techniques.
Acknowledgments
We would like to thank René Nyffenegger from the Institute of Applied Physics of the
University of Bern for his valuable contribution to the experimental work. This research was
supported by the Swiss National Science Foundation (Grant No. 205320-127274).

Determining The Optical Properties of Gelatine Gel

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Determining The Optical Properties of Gelatine Gel

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Determining the optical properties of a

gelatin-TiO2 phantom at 780 nm

Abstract: Tissue phantoms play a central role in validating biomedical

References and links

3.2. Diffusion model

3.4. Results and discussion

Parameters Phantom Glass Aquarium Air

6.1. Testing the diffusion limit, influence of scattering law

Transport mean Absorption length

* 4.5(±0.5) mm [ µe′ ≈ µ s′= (0.22 ± 0.02) mm −1 ];

Absorption length:  a = 190(+10 − 20) mm [ µ a = 0.0053(+0.0006 − 0.0003) mm -1 ];

Extinction coefficient: µe ≈ µ s = 2.86(±0.04) mm -1 .

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