The optical properties of rat
abdominal wall muscle
Luís Oliveira1,2,3, Maria Inês Carvalho4, Elisabete Nogueira1,3,
Valery Tuchin5,6,7
1
Physics Department – Polytechnic of Porto, School of Engineering, Rua Dr. António Bernardino de
Almeida, 431, 4200-072 Porto, Portugal.
2
3 CIETI
FEUP – University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal.
– Centre of Innovation in Engineering and Industrial Technology, ISEP, Rua Dr. António
Bernardino de Almeida, 431, 4200-072 Porto, Portugal.
4 DEEC/FEUP
and INESC TEC, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal.
5 Research-Educational
6 Laboratory
Institute of Optics and Biophotonics, Saratov State University, 83 Astrakhanskaya
Str., Saratov 410012, Russia.
of Laser Diagnostics of Technical and Living Systems, Institute of Precise Mechanics and
Control RAS, Saratov, 410028, Russia.
7 Optoelectronics
and Measurement Techniques Laboratory, University of Oulu, PO Box 4500 FI90014, Oulu, Finland.
The
optical
properties
of rat
abdominal
wall
muscle
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
1
Saratov Fall Meeting
2013
1. Introduction
The knowledge of the optical properties of biological
tissue and their wavelength dependency is very
important for clinical applications and related research
where optical technologies are to be used [1][2].
The basic optical properties are the absorption
coefficient (µa), the scattering coefficient (µs), the
reduced scattering coefficient (µs’) and the anisotropy
factor (g) [3].
The absorption coefficient quantifies the amount of
absorbed photons per unit length inside the biological
tissue [4].
The
optical
properties
of rat
abdominal
wall
muscle
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
2
Saratov Fall Meeting
2013
The scattering coefficient quantifies the amount of
scattered photons per unit length inside the biological
tissue [4].
The reduced scattering coefficient quantifies both the
amount of scattered photons and directionality of
scattering per unit length inside the biological tissue [4].
The anisotropy factor quantifies the directionality of the
scattering events inside the biological tissue [4].
The determination of such optical properties can be
made using direct and indirect methods, depending on
the instrumentation available and desired precision [3].
The
optical
properties
of rat
abdominal
wall
muscle
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
3
Saratov Fall Meeting
2013
Among the indirect methods, computer simulations
using the Adding-Doubling and Monte Carlo methods
are commonly used and generated results present high
precision [3].
Some computer codes based on these methods are
available. They are used to perform inverse simulations
and estimate the optical properties of biological tissues
[5] [6] [7] [8].
The abdominal wall muscle from rat is a fibrous tissue
that contains a collection of fiber bundles distributed
over the interstitial fluid [9].
The muscle fiber bundles contain several muscle fibers,
which are chains of proteins (actin and myosin) [10].
The
optical
properties
of rat
abdominal
wall
muscle
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
4
Saratov Fall Meeting
2013
The interstitial fluid is a background liquid that contains
mainly water and some dissolved salts and minerals [10].
Due to the composition of the muscle, we expect to
observe much higher values for the scattering
coefficient than for the absorption coefficient [3].
Such fact is due to the refractive index profile observed
between the scatterers (muscle fibers) and the
background material (interstitial fluid) [10].
To study the optical properties of the abdominal wall
muscle from rat – species Wistar Han, we have adopted
an indirect technique and estimated the optical
properties from inverse simulations using the Monte
Carlo and the Adding Doubling codes.
The
optical
properties
of rat
abdominal
wall
muscle
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
5
Saratov Fall Meeting
2013
We have used some experimental assemblies to measure
transmittance and reflectance spectra from muscle
samples. Afterwards, we have retrieved experimental
values from these spectra between 400 and 1000 nm to
use as input in the inverse simulations [9].
As we have observed with this study, our experimental
measurements present errors that induce bad wavelength
dependencies for the estimated optical properties.
By comparing the wavelength dependencies obtained
from our study with the ones published in literature
[11][12], we could correct our results and identify the
source of those errors in the experimental set-ups that
we have used.
The
optical
properties
of rat
abdominal
wall
muscle
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
6
Saratov Fall Meeting
2013
2. Experimental Methodology
We have developed an inverse MC simulation code based
on the code developed for the forward problem. Such
forward code is designated as Monte Carlo for MultiLayered media (MCML) and was developed by Lihong
Wang and Steven Jacques in 1992 [5].
Such forward code is freeware and can be found on the
internet, at: http://omlc.ogi.edu/software/mc/.
Similarly, we have also used an IAD calculator developed
by Scott Prahl [6]. This calculator was used to estimate the
optical properties of the muscle. The calculator can be
found at: http://omlc.ogi.edu/calc/iad_calc.html.
The
optical
properties
of rat
abdominal
wall
muscle
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
7
Saratov Fall Meeting
2013
Based on the input parameters for the IMC code and the
IAD calculator, we have chosen to perform experimental
measurements of Total transmittance (Tt), Total reflectance
(Rt), Collimated transmittance (Tc) and Specular
reflectance (Rs).
The measurements of Tt and Rt were made using an
integrating sphere. The measuring set-ups used to measure
Tc and Rs were specially constructed for this study.
In the Tc measuring set-up we have used a light beam from
a tungsten-halogen lamp to illuminate the sample – see
figure 1:
The
optical
properties
of rat
abdominal
wall
muscle
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
8
Saratov Fall Meeting
2013
Figure 1: Tc measuring set-up.
The
optical
properties
of rat
abdominal
wall
muscle
Figure 2 shows the experimental set-up used to
measure Rs. The 8º angle used to illuminate the sample
and to detect the reflected beam is accordingly to the
illumination angle used in the measurement of Rt.
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
9
Saratov Fall Meeting
2013
Figure 2: Rs measuring set-up.
The
optical
properties
of rat
abdominal
wall
muscle
From the spectra measured with each experimental
assembly, we considered all wavelengths separated by
25 nm between 400 and 1000 nm.
The measurements of Tt, Rt and Ts are sufficient to
use in the IAD calculator.
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
To perform the inverse MC simulations, we need the
same measurements as used for IAD simulations.
Additionally, IMC code needs the absorbance (A)
and the diffuse reflectance (Rd).
10
We will explain below how to calculate both A and Rd
from the other measurements.
Saratov Fall Meeting
2013
The
optical
properties
of rat
abdominal
wall
muscle
3. Experimental results
Using the experimental set-ups represented in figures 1 and 2
and also the ones to obtain the integrated measurements, we
have measured the different spectra of natural rat muscle.
Figure 3 presents these spectra:
(a)
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
11
Saratov Fall Meeting
2013
(b)
(d)
(c)
Figure 3: Measured spectra from natural muscle: (a) Tt, (b) Tc,
(c) Rt and (d) Rs.
The
optical
properties
of rat
abdominal
wall
muscle
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
4. Computer simulations
To perform the inverse MC simulations to generate the
optical properties of the muscle, we needed to calculate
the natural spectra for Rd and A.
Using the integrated measurements (Tt and Rt), we have
calculated the A spectrum as [9]:
A 1  Tt  Rt 
(1)
Using the Rt and Rs spectra we have calculated the Rd
spectrum as the difference [9]:
12
Saratov Fall Meeting
2013
R d  Rt  Rs
(2)
The
optical
properties
of rat
abdominal
wall
muscle
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
13
Saratov Fall Meeting
2013
The A and Rd spectra calculated are presented in
figure 4:
Figure 4: Calculated spectra: A – left; Rd – right.
By considering three sets of measurements and
calculations, as described above, we have performed
three sets of inverse simulations using our IMC code
and other three sets of simulations using the IAD
calculator.
The
optical
properties
of rat
µ and µ
abdominal
present
reasonable
wall
smooth
wavelength
muscle
a
We have calculated the mean optical properties for natural
muscle, both from the results of the IMC and IAD
simulations. Figure 5 presents both mean results:
s
dependence.
Luís Oliveira
Maria Inês Carvalho
µs’ andNogueira
g
Elisabete
present
non
Valery Tuchin
monotonic
behavior [11]
[12].
14
Saratov Fall Meeting
2013
Figure 5: Mean optical properties for rat muscle between 400 and 1000 nm.
Results from three studies using IMC (blue) and using IAD (green).
The
optical
properties
of rat
abdominal
wall
muscle
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
15
Saratov Fall Meeting
2013
5. Corrections to wavelength
dependency
The non monotonic behavior seen for µs’ and g on
figure 5 are a result of erroneous measurements for Tc
and Rs.
By considering the experimental set-up to measure Tc
(see figure 1), more than unscattered transmitted
photons are detected. Considering the Rs set-up
presented in figure 2, we see that a great amount of
diffused reflected photons are also collected by the
detector [13].
Such errors in our measurement set-ups produce wrong
spectra that will generate erroneous optical properties.
Such errors are more evident for lower wavelengths.
The
optical
properties
of rat
abdominal
wall
muscle
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
16
Saratov Fall Meeting
2013
To correct these errors, we have considered the typical
wavelength dependencies for µs and µs’ that are
presented in literature [11] [12].
We have observed that for the case of µa, we have
similar wavelength dependency as indicated in literature
[11] [14].
In the case of µs, our results show also a wavelength
dependence that is similar to typical behavior seen in
literature [11] [14].
The major difference between our results and literature
is seen for µs’ [11] [12]. In this case, to correct our results,
we had to neglect the values obtained for wavelengths
between 450 and 650 nm and then adjust the remaining
values with an exponential decay.
The
optical
properties
of rat
abdominal
wall
muscle
After calculating the mean optical properties from the
ones obtained from IMC and IAD, we have represented in
figure 6 the cases of µa, µs and µs’. In the cases of µs and
µs’ we have also represented the modeling adjustment
lines and corresponding equations for the wavelength
range considered.
 s  3.129 *106 1.927  84.4
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
 s '  5.104 * 105 1.763  6.941
17
Saratov Fall Meeting
2013
Figure 6: Mean µa, mean µs and
mean µs’, with error bars and
wavelength dependence according
to literature.
After obtaining the wavelength dependencies for µa, µs
and µs’, we have calculated the smooth wavelength
dependence for the g-factor as 1-µs’/µs. Figure 7 presents
such wavelength dependence:
Mean smooth data
considering IMC and IAD simulations
0.9
0.89
0.88
0.87
g
The
optical
properties
of rat
abdominal
wall
muscle
0.86
0.85
0.84
0.83
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
18
Saratov Fall Meeting
2013
0.82
400
500
600
700
800
Wavelength (nm)
900
1000
Figure 7: wavelength dependence for the g-factor of rat
muscle.
The smooth wavelength dependencies seen in graphs of
figures 6 and 7 represent the correct optical properties
of the rat muscle.
The
optical
properties
of rat
abdominal
wall
muscle
Figures 8 and 9 show the spectra from our experimental
measurements and the spectra generated from the
optical properties presented in figures 6 and 7, for
comparison.
0.7
0.6
Tt (A.U.)
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
Using these optical properties, we have performed
forward MC simulations to generate the optical
measurements that should be obtained from the muscle
sample if they were made correctly.
0.5
0.4
Experimental
Generated
0.3
19
Saratov Fall Meeting
2013
0.2
400
500
600
700
800
Wavelength (nm)
900
1000
Figure 8: Transmittance measurements: (a) Tt and (b) Tc.
0.24
Experimental
Generated
0.03
0.028
0.22
Rs (A.U.)
0.026
Rt (A.U.)
The
optical
properties
of rat
abdominal
wall
muscle
0.032
0.2
0.18
0.024
0.022
0.02
Experimental
Generated
0.16
0.016
0.14
400
0.018
500
600
700
800
Wavelength (nm)
900
1000
0.014
400
500
600
700
800
Wavelength (nm)
900
1000
Figure 9: Reflectance measurements: (a) Rt and (b) Rs.
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
Observing figures 8 and 9, we see that the most significant
difference between experimental and generated spectra is
verified for the case of Rs. Such difference indicates that our
experimental set-up to measure Rs implies the combined
measurement of Rs and Rd light.
20
The differences seen for Tt, Tc and Rt measurements are not
too significant – Integrated measurements present
differences below 2% and Tc even less.
Saratov Fall Meeting
2013
The
optical
properties
of rat
abdominal
wall
muscle
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
21
Saratov Fall Meeting
2013
6. Conclusions
With the research that we have developed, we have obtained the
optical properties for skeletal muscle of the Wistar Han rat for
the wavelength range of 400 to 1000 nm.
Such optical properties were obtained not only from optical
measurements. We needed to model the wavelength dependence
of μs and μs’, accordingly to what is presented in literature.
By performing such modeling of wavelength dependece for the
scattering and reduced scattering coefficients, we were abble to
identify errors in our experimental measurements.
The knowledge of the optical properties of natural rat muscle
allow us to develop or improve clinical applications for the
muscle in the wavelength range of 400 to 1000 nm.
The
optical
properties
of rat
abdominal
wall
muscle
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
22
Saratov Fall Meeting
2013
Another application that can make use of the optical properties
that we have estimated concerns the study of optical clearing of
the muscle.
With the optical properties for the natural muscle, we can now
calculate their variations and also estimate the variations in the
refractive index of the tissue under treatment with an optical
clearing agent.
Such is in fact a great motivation to proceed with our research
and we expect to confirm that the optical clearing treatment
produces a decrease in the scattering coefficient as indicated in
literature [15].
The
optical
properties
of rat
abdominal
wall
muscle
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
23
Saratov Fall Meeting
2013
Acknowledgements
The authors would like to acknowledge the help given in the
preparation of tissue samples by LAIMM – Laboratório de Apoio
à Investigação em Medicina Molecular, Departamento de
Biologia Experimental, Faculdade de Medicina da Universidade
do Porto, Portugal.
For the resources and instruments made available to perform the
present research, the authors would also like to thank CIETI –
Centre of Innovation in Engineering and Industrial Technology
and the Physics Department of Polytechnic of Porto – School of
Engineering, Portugal.
VTT was supported by Russian Federation President’s grant
“Scientific Schools,”1177.2012.2; FiDiPro, TEKES Program
(40111/11), Finland; SCOPES EC, Uzb/Switz/RF, Swiss NSF,
IZ74ZO_137423/1; RFBR-13-02-91176-NSFC_a; RF Governmental
contracts 14.B37.21.0728, 14.B37.11.0563, and 14.512.11.0022
The
optical
properties
of rat
abdominal
wall
muscle
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
24
Saratov Fall Meeting
2013
References
[1] A. N. Bashkatov, E. A. Genina, V. I. Kochubey, V. V. Tuchin,
“Optical properties of human skin, subcutaneous and mucous
tissues in the wavelength range from 400 to 2000 nm”, J. Phys.
D: Appl Phys, 38, pp. 2543 – 2555, (2005).
[2] R. Cicchi, F. S. Pavone, D. Massi, D. D. Sampson, “Contrast
and Depth enhancement in two-photon microscopy of human
skin ex vivo by use of optical clearing agents”, Optics Express
13(7), (2005).
[3] V. V. Tuchin, “Tissue Optics: Light Scattering Methods and
Instruments for Medical Diagnosis”, 2nd ed., SPIE Press,
Bellingham, 2007.
[4] Tuan Vo-Dinh, “Biomedical Photonics Handbook”, CRC Press
LLC, 2003.
The
optical
properties
of rat
abdominal
wall
muscle
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
25
Saratov Fall Meeting
2013
[5] L.-H. Wang, S. L. Jacques, L.-Q. Zheng, “MCML – Monte
Carlo modeling of photon transport in multi-layered tissues”,
Computer Methods and Programs in Biomedicine, 47, 131 –
146, 1995.
[6] S. A. Prahl. “The adding-doubling method”. In A. J. Welch and
M. J. C. van Gemert, editors, Optical-Thermal Response of
Laser Irradiated Tissue, chapter 5, pages 101-129. Plenum
Press, 1995.
[7] V. V. Tuchin, “Tissue Optics: Light Scattering Methods and
Instruments for Medical Diagnosis”, 2nd ed., SPIE Press,
Bellingham, 2007.
[8] [8] E. Zamora-Rojas, B. Aernouts, A. Garrido-Varo, D. PérezMarín, J. E. Guerrero-Ginel, W. Saeys, “Double integrating
sphere measurements for estimating optical properties of pig
subcutaneous adipose tissue”, Innovative Food Science and
Emerging Technologies, 19, pp. 218-226, (2013).
The
optical
properties
of rat
abdominal
wall
muscle
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
26
Saratov Fall Meeting
2013
[9] L. M. Oliveira, M. I. Carvalho, E. M. Nogueira, V. V. Tuchin,
“Optical measurements of rat muscle samples under treatment
with ethylene glycol and glucose”, Journal of Innovative
Optical Health Sciences, 6(2), 1350012-1 – 1350012-15,
(2013).
[10] L. M. Oliveira, M. I. Carvalho, E. M. Nogueira, V. V. Tuchin,
“The characteristic time of glucose diffusion measured for
muscle tissue at optical clearing”, Laser Phys. 23(7), 075606
(6pp), 2013.
[11] S. L. Jacques, “Optical properties of biological tissues: a
review”, Phys. Med. Biol. 58, pp. R37 – R61, (2013).
[12] A. N. Bashkatov, E. A. Genina, V. V. Tuchin, “Optical
properties of skin, subcutaneous, and muscle tissues: a review”,
Journal of Innovative Optical Health Sciences, 4(1), pp. 9 – 38,
2011.
The
optical
properties
of rat
abdominal
wall
muscle
Luís Oliveira
Maria Inês Carvalho
Elisabete Nogueira
Valery Tuchin
27
Saratov Fall Meeting
2013
[13] E. Vitkin, V. Turzhitsky, L. Qiu, L. Guo, I. Itzkan, E. B.
Hanlon, L. T. Perelman, “Photon diffusion near the point-ofentry in anisotropically scattering turbid media”, Nature
communications, 2011. Corrigir depois de ver citação na net.
[14] A. N. Bashkatov, E. A. Genina, V. I. Kochubey, A. A.
Gavrilova, S. V. Kapralov, V. A. Grishaev, V. V. Tuchin,
“Optical properties of human stomach mucosa in the spectral
range from 400 to 2000 nm: prognosis for gastroenterology”,
Medical Laser Applications, 22, pp. 95 – 104, (2007).
[15] V. V. Tuchin, “Optical Clearing of Tissues and Blood”, SPIE
Press, Bellingham, WA (2006).