Evaluation of a fiberoptic-based system for measurement of optical properties in highly attenuating turbid media.

Abstract

Accurate measurements of the optical properties of biological tissue in the ultraviolet A and short visible wavelengths are needed to achieve a quantitative understanding of novel optical diagnostic devices. Currently, there is minimal information on optical property measurement approaches that are appropriate for in vivo measurements in highly absorbing and scattering tissues. We describe a novel fiberoptic-based reflectance system for measurement of optical properties in highly attenuating turbid media and provide an extensive in vitro evaluation of its accuracy. The influence of collecting reflectance at the illumination fiber on estimation accuracy is also investigated.
A neural network algorithm and reflectance distributions from Monte Carlo simulations were used to generate predictive models based on the two geometries. Absolute measurements of diffuse reflectance were enabled through calibration of the reflectance system. Spatially-resolved reflectance distributions were measured in tissue phantoms at 405 nm for absorption coefficients (mu(a)) from 1 to 25 cm-1 and reduced scattering coefficients (mu'(s)) from 5 to 25 cm-1. These data and predictive models were used to estimate the optical properties of tissue-simulating phantoms.
By comparing predicted and known optical properties, the average errors for mu(a) and mu'(s) were found to be 3.0% and 4.6%, respectively, for a linear probe approach. When bifurcated probe data was included and samples with mu(a) values less than 5 cm-1 were excluded, predictive errors for mu(a) and mu'(s) were further reduced to 1.8% and 3.5%.
Improvements in system design have led to significant reductions in optical property estimation error. While the incorporation of a bifurcated illumination fiber shows promise for improving the accuracy of mu's estimates, further study of this approach is needed to elucidate the source of discrepancies between measurements and simulation results at low mu(a) values.

Full text

Page 1

BioMed Central
Page 1 of 14
(page number not for citation purposes)
BioMedical Engineering OnLine
Open Access
Research
Evaluation of a fiberoptic-based system for measurement of optical
properties in highly attenuating turbid media
Divyesh Sharma, Anant Agrawal, L Stephanie Matchette and T Joshua Pfefer*
Address: Food and Drug Administration, Center for Devices and Radiological Health, Rockville, Maryland, USA
Email: Divyesh Sharma - divyesh_sharma2001@yahoo.com; Anant Agrawal - anant.agrawal@fda.hhs.gov; L
Stephanie Matchette - stephanie.matchette@fda.hhs.gov; T Joshua Pfefer* - joshua.pfefer@fda.hhs.gov
* Corresponding author
Abstract
Background: Accurate measurements of the optical properties of biological tissue in the ultraviolet A and short visible
wavelengths are needed to achieve a quantitative understanding of novel optical diagnostic devices. Currently, there is minimal
information on optical property measurement approaches that are appropriate for in vivo measurements in highly absorbing and
scattering tissues. We describe a novel fiberoptic-based reflectance system for measurement of optical properties in highly
attenuating turbid media and provide an extensive in vitro evaluation of its accuracy. The influence of collecting reflectance at the
illumination fiber on estimation accuracy is also investigated.
Methods: A neural network algorithm and reflectance distributions from Monte Carlo simulations were used to generate
predictive models based on the two geometries. Absolute measurements of diffuse reflectance were enabled through calibration
of the reflectance system. Spatially-resolved reflectance distributions were measured in tissue phantoms at 405 nm for
absorption coefficients (µa) from 1 to 25 cm-1 and reduced scattering coefficients (
predictive models were used to estimate the optical properties of tissue-simulating phantoms.
) from 5 to 25 cm-1. These data and
Results: By comparing predicted and known optical properties, the average errors for µa and
4.6%, respectively, for a linear probe approach. When bifurcated probe data was included and samples with µa values less than
5 cm-1 were excluded, predictive errors for µa and were further reduced to 1.8% and 3.5%.
′ µs
were found to be 3.0% and
Conclusion: Improvements in system design have led to significant reductions in optical property estimation error. While the
incorporation of a bifurcated illumination fiber shows promise for improving the accuracy of
approach is needed to elucidate the source of discrepancies between measurements and simulation results at low µa values.
estimates, further study of this
Background
In recent years, advances in optical technology have
helped facilitate rapid progress in fluorescence and reflect-
ance spectroscopy-based techniques for medical diagnos-
tics. Recent studies involving ultraviolet A (UVA, 320 to
400 nm) and shorter visible (VIS) wavelengths (400 to
550 nm) have demonstrated that spectroscopic
approaches can be highly effective for a variety of applica-
tions including intravascular detection of atherosclerotic
plaque [1], in situ brain tumor demarcation [2] and sur-
veillance for neoplasia in mucosal tissues that line organs
such as the lungs and cervix [3]. While clinical studies
Published: 23 August 2006
BioMedical Engineering OnLine 2006, 5:49doi:10.1186/1475-925X-5-49
Received: 04 April 2006
Accepted: 23 August 2006
This article is available from: http://www.biomedical-engineering-online.com/content/5/1/49
© 2006 Sharma et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
′ µs
′ µs
′ µs

Page 2

BioMedical Engineering OnLine 2006, 5:49http://www.biomedical-engineering-online.com/content/5/1/49
Page 2 of 14
(page number not for citation purposes)
have shown significant promise, further improvements in
efficacy are needed for this technology to achieve its full
potential.
Accurate approaches for in vivo measurement of tissue
optical properties in UVA and short VIS wavelengths are
needed to optimize the ability of diagnostic devices. Since
some tissue discrimination algorithms [4] use optical
property data as inputs, the accuracy of this data may
directly influence a system's diagnostic efficacy. Further-
more, numerical and analytical models are effective tools
for elucidating light-tissue interaction phenomena and
identifying optimal device designs, including selection of
excitation and collection wavelengths and probe geome-
try [5,6]. However, the accuracy of input parameters such
as tissue optical properties can strongly influence the
quality and usefulness of modeling results.
The most important optical properties for describing light
propagation in tissue are absorption coefficient (µa) and
reduced scattering coefficient {
convenient combination of the scattering coefficient (µs)
and the scattering phase function (g). While prior optical
property studies have typically involved VIS to near-infra-
red wavelengths where tissue tends to have relatively low
attenuation levels [7], data on in vivo optical properties in
the UVA and lower VIS wavelength ranges are limited.
This is likely due in part to the fact that many earlier stud-
ies were performed in support of photon migration appli-
cations. Another factor may have been the difficulties
associated with measuring signals in tissue with high
attenuation levels, such as the rapid decay in reflected
light levels with distance from the source location.
= µs(1-g)}, which is a
In spite of the limited literature on optical property meas-
urements at relevant UVA and short VIS wavelengths,
prior studies at longer wavelengths have produced signif-
icant advances in experimental, computational and ana-
lytical techniques, some of which are applicable in our
spectral range of interest. While time-[8] and frequency-
[9] domain approaches have shown promise at long VIS
and near-infrared wavelengths, these approaches tend to
be expensive and have not shown efficacy at shorter wave-
lengths. Spatially-resolved approaches typically involve
using tissue phantoms or computer simulations to gener-
ate data for inverse model calibration, and then using the
model to estimate optical properties based on reflectance
measurements [10]. In prior studies, diffuse reflectance
has been collected from tissue phantoms over a broad
range of optical properties and wavelengths [10-12].
Monte Carlo [10,11] or diffusion approximation [13]
light propagation models are typically used to generate
spatially-resolved reflectance profiles, although the latter
are limited to larger source-detector separation distances
which satisfy diffusion criteria. Reflectance measurements
are typically performed with multiple-channel fiberoptic
bundles, or probes, which deliver light to the tissue sur-
face and collect reflectance at two or more well-defined
distances from the source fiber. Optical fibers are com-
monly arranged in either circular [11-14] or linear [15,16]
patterns on the face of the fiberoptic probe. The source-
collection separation distances used in prior studies have
ranged from several millimeters to centimeters [11,14-
18]. Properties such as numerical aperture (NA) and fiber
diameter have varied widely in prior studies. One recent
study involved an approach based on bifurcated probes of
varying aperture diameter, rather than the more typical
linear array approach [19]. Multivariate calibration tech-
niques such as neural networks (NN) [10,13,20], fuzzy
logic [21], regression [11] and partial least squares [22]
have been used to solve the inverse problem of determin-
ing optical properties from reflectance.
In a prior pilot study, we evaluated several different
approaches to fiberoptic probe-based optical property
determination using Monte Carlo simulations and diffuse
reflectance measurements [23]. By calibrating a neural
network model with simulated reflectance data based on
uniformly distributed optical properties and validating
the model with measurements of tissue phantoms having
randomly distributed optical properties, moderately high
levels of accuracy were found: root mean square errors of
1.58 cm-1 for µa and 2.35 cm-1 for
late to average predictive errors of 12.5% and 16.2%.
Optical property estimation errors may be attributed in
part to the measurement approach in which large varia-
tions in intensity from fiber-to-fiber were produced at the
CCD camera, as well as the limited dynamic range (14-
bit) and noise levels generated during long exposures. In
general, these results were sufficient for a pilot study, yet
they indicated that refinements would be needed before
accurate in vivo measurements could be achieved.
. These values trans-
In an effort to improve upon the results of the pilot study
and develop a system that is sufficiently accurate to pro-
vide meaningful data during in vivo measurements, signif-
icant modifications were made to the prior system. The
goals of the current study were to evaluate the accuracy of
this second generation optical property measurement sys-
tem and assess the potential added benefit of a bifurcated
fiberoptic probe that enables detection of reflectance from
the illumination site.
′ µs
′ µs

Page 3

BioMedical Engineering OnLine 2006, 5:49 http://www.biomedical-engineering-online.com/content/5/1/49
Page 3 of 14
(page number not for citation purposes)
Methods
System description
Diagrams of the experimental setup and fiberoptic probe
face used to perform diffuse reflectance measurements are
presented in Figure 1. The source is a 405 nm diode laser
(LCDU 12/5431, Power Technology, Inc; Little Rock, AR)
with a power level of 2.5 mW. The input power is adjusted
using neutral density (ND) filters (CVI Laser Corporation,
Albuquerque, NM). The custom designed fiberoptic probe
(FiberTech Optica, Ontario, Canada) is used to deliver
laser light from the source to the sample and guide diffuse
reflectance from the sample to the detector. The diameter
of the probe face is 0.5 cm which would make it practical
for in vivo studies of internal organ sites. The probe con-
tains a single illumination fiber and five detection fibers
spaced at consecutive center-to-center distances of 0.5
mm. The core diameter of each fiber is 0.2 mm with a NA
of 0.22. The detection legs are connected to five in-line fil-
ters (FHS-UV, Ocean Optics, Dunedin, FL) three of which
contained ND filters. The filter holders are coupled to the
legs of a second probe, the common end of which con-
tains a linear fiber array that was aligned to the entrance
slit of an imaging spectrometer (SpectraPro 300i, Acton
Research Corp., Acton, MA). The output of the spectrom-
eter is detected by a low noise, high dynamic range (16-
bit) CCD camera (Princeton Instruments Spec10:400B,
Trenton, New Jersey) and acquired using proprietary soft-
ware (WinSpec Princeton Instruments, Trenton, NJ).
System Diagram
Figure 1
System Diagram. Diagram includes (a) experimental setup and (b) fiberoptic probe face.





405 nm



















D = 0.5 cm
Diode Laser
Illumination
Fiber
Sample

Detection
Fibers

In-line
ND Filters
Spectrometer and
CCD Camera

Computer with Image
Acquisition Software

(a)
(b)

Page 4

BioMedical Engineering OnLine 2006, 5:49http://www.biomedical-engineering-online.com/content/5/1/49
Page 4 of 14
(page number not for citation purposes)
Experimental system modifications
In our previous system, the large range of light levels (Fig-
ure 2) incident on the detector necessitated measurements
at multiple exposure durations to acquire a single reflect-
ance distribution. By improving the homogeneity of light
levels delivered by the fiberoptic probe, a single CCD
acquisition could record all fiber intensities. This was
achieved by using in-line ND filters [24] for each fiber. By
analyzing the detected signal levels for four samples at the
edges of the designated optical property parameter space
(µa = 1, 25 cm-1, = 5, 25 cm-1) we identified a combi-
nation of ND filters for each fiber which would produce
moderately high output levels on the CCD without induc-
ing saturation. Combined attenuation levels of 2.2, 1.9,
and 0.3 OD were used for the fibers at separation dis-
tances of 0.5, 1.0 and 2.0 mm. The ND levels chosen for
these fibers appear irregular due to variations in fiber
transmittance and in coupling efficiency of the in-line fil-
ters. Thus the system was optimized in such a way that a
single set of ND filters could be used to collect reflectance
for in vitro or in vivo samples having optical properties
anywhere within the relevant range (µa = 1–25 cm-1,
5–25 cm-1). Figure 2 shows computational modeling
results for a sample with µa = 25 cm-1,
order to illustrate the wide range of intensities that must
be detected. This graph also includes a measurement of a
tissue phantom with the same optical properties, indicat-
ing that the variation of intensity at the CCD has been
reduced from five orders of magnitude to two through the
use of ND filters. The exposure duration required for
acquiring sufficient signal ranged from 0.12 to 25 seconds
depending on the sample attenuation level.
=
= 15 cm-1 in
The second optimization task involved enabling absolute
measurements by calibrating the measured intensity to
simulation results. In order to accomplish this goal, CCD
measurements for all detection fibers were made at two
different illumination intensities in three different phan-
toms : µa = 1 cm-1 and = 5 cm-1, µa = 1 cm-1 and
25 cm-1 and µa = 2 cm-1 and
position, the relationship between measured CCD counts
and Monte Carlo-predicted intensity levels were graphed
=
= 25 cm-1. For each fiber
′ µs
′ µs
′ µs
′ µs
′ µs
′ µs
Reduction of detected signal range
Figure 2
Reduction of detected signal range. Monte Carlo simulations and experimental data for µa = 25 cm-1,
signal range in the experimental data was reduced by using neutral density filters to preferentially attenuate the channels clos-
est to the source fiber.
= 15 cm-1. The
1.0E+00
1.0E+01
1.0E+02
1.0E+03
1.0E+04
1.0E+05
1.0E+06
00.511.522.53
Distance From Center of Illumination Fiber (mm)
Measured Reflectance (mm-2)
1.0E-09
1.0E-08
1.0E-07
1.0E-06
1.0E-05
1.0E-04
1.0E-03
Simulated Reflectance (AU)
Simulated Intensity
Measured Reflectance

Simulated Reflectance
Measured Intensity (w/ ND filters)
µ µ µ µa=25 cm-1, µ µ µ µs'=15 cm-1
′ µs

Page 5

BioMedical Engineering OnLine 2006, 5:49http://www.biomedical-engineering-online.com/content/5/1/49
Page 5 of 14
(page number not for citation purposes)
for the two phantoms. A linear fit to these points and the
origin was calculated for each fiber. These linear fits were
used as calibration equations to convert CCD counts to
absolute intensity levels for all measurements during this
study.
Bifurcated fiberoptic probe measurements
The objective of this component of the study was to eval-
uate the utility of adding a bifurcated illumination fiber to
the linear-array geometry. This work was performed for
two primary reasons: (1) computational results that indi-
cate that at positions near the illumination fiber there is a
relatively high level of variation in reflectance intensity as
changes [23]; and (2) a prior investigation has indi-
cated that a sized-fiber approach involving collecting
reflectance at the site of illumination is highly effective for
determining tissue optical properties [19]. A bifurcated
fiberoptic probe (Innova Quartz, Phoenix, AZ) – illumi-
nation and collection fibers fused to a third common fiber
which contacted the sample – with a core diameter of 0.2
mm and NA of 0.22 was used to perform the measure-
ments (Figure 3) [25]. Light collected from the detection
leg of the probe was measured by a power meter (Newport
Model 1930C, 818-ST-UV detector, Irvine, CA) for each of
the 60 samples previously used for the linear array meas-
urements.
The reflectance collected at the illumination site of the
sample is the total reflectance (Rtotal) which consists of
two components: diffuse reflectance (Rdiffuse) and specular
reflectance (Rspecular). Fresnel reflections for both fiber
core/air and fiber core/water were measured. The probe
geometry resulted in multiple reflections at the bifurca-
tion point. Thus, baseline reflectance measurement was
made using fiber core refractive index matching liquid (n
= 1.47, Cargille Laboratories, Inc; Cedar Grove, NJ). Rdiffuse
was obtained by normalizing all the data points to the
Fresnel reflections for the fiber core/water as shown
below:
Rdiffuse = Rtotal - Rspecular (1)
Where,
Psample = Power measured from the sample
Pη = Power measured from the index matching liquid
Pwater = Power output from fiber core/water
Pinput = Power output from the probe
E.F. = Fiber efficiency factor obtained for the collection
fiber
Experimentally measured Fresnel reflectance was within
10% of the theoretical value for the fiber core/water inter-
face. The fiber efficiency factor was obtained by coupling
light from a 100 µm core diameter fiber into the common
end of the bifurcated fiber and measuring the output from
the detection leg of the probe. Measurements were made
on the same sets of phantoms used for linear-array fiber
system.
Tissue phantoms
Since the validity of the system evaluation is dependent
on the accuracy of tissue phantom optical properties, eval-
uation of phantom materials and benchmarking of phan-
toms was performed at the outset of this study. The optical
properties of individual tissue phantoms were determined
using an inverse adding-doubling approach [26] and a
spectrophotometer (Shimadzu UV-3101PC, Columbia,
MD). This data was compared to theoretical estimates
based on Mie theory and direct collimated absorption
measurements of the scatterer and absorber, respectively.
′ µs
R
PP
E F
. .
P
total
sample
input
=
−
()
∗
( )
2
η
R
PP
E F
. .
P
specular
water
input
=
−
()
∗
( )
3
η
Diagram of bifurcated probe
Figure 3
Diagram of bifurcated probe. A bifurcated fiberoptic
probe was used to measure reflectance at the illumination
site.
Illumination
Fiber
Common
Fiber
Detection
Fiber
Sample