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In vivo determination of skin near-infrared optical properties using
diffuse optical spectroscopy
Sheng-Hao Tseng, Alexander Grant, and Anthony J. Durkin
University of California, Irvine Beckman Laser Institute Laser Microbeam and Medical Program
Irvine, California 92617 E-mail: adurkin@uci.edu
Abstract
We develop a superficial diffusing probe with a 3mm source-detector separation that can be used in
combination with diffuse optical spectroscopic (DOS) methods to noninvasively determine full-
spectrum optical properties of superficial in vivo skin in the wavelength range from 650 to 1000 nm.
This new probe uses a highly scattering layer to diffuse photons emitted from a collimated light
source and relies on a two-layer diffusion model to determine tissue absorption coefficient μ
a
and
reduced scattering coefficient
. By employing the probe to measure two-layer phantoms that mimic
the optical properties of skin, we demonstrate that the probe has an interrogation depth of 1 to 2mm.
We carry out SSFDPM (steady state frequency-domain photon migration) measurements using this
new probe on the volar forearm and palm of 15 subjects, including five subjects of African descent,
five Asians, and five Caucasians. The optical properties of in vivo skin determined using the
superficial diffusing probe show considerable similarity to published optical properties of carefully
prepared ex vivo epidermis+dermis.
Keywords
tissue optics; optical properties; epithelial tissue; near-infrared spectroscopy; photon migration
1 Introduction
The ability to accurately quantify the optical properties of superficial skin is important for many
clinical applications including the determination of light distribution in laser-induced
cutaneous therapies;
1
monitoring skin blood oxygenation, melanin concentration,
2
and water
concentration;
3
and improving noninvasive optical measurements of tissue.
4
Investigation of
the optical properties of skin has been carried out by several researchers. Troy and Thennadil,
5
Simpson et al.,
6
and Baskatov et al.,
7
characterized the optical properties of ex vivo skin
using an integrating-sphere-based approach. Although the optical properties of skin can be
assessed using such ex vivo techniques, the necessity of taking biopsies from subjects’ skin
limits the clinical usefulness of these techniques. Currently, in vivo techniques that are capable
of measuring optical properties of skin volumes consisting primarily of epidermis+dermis do
exist, but are limited. For example, Zhang et al. determined the optical properties of in vivo
skin using visible reflectance spectroscopy along with a multilayer skin model and a genetic
optimization algorithm.
8
A multilayer skin model and a number of fitting parameters, such as
layer thickness, chromophores, and scattering properties for each layer, and their corresponding
ranges must be chosen carefully in advance to avoid nonuniqueness in the solution space.
Address all correspondence to Sheng-Hao Tseng, Johnson&Johnson CPPW, 199 Grandview Rd. - Skillman, NJ 08558; Tel.: 609–269–
5263; Fax: 609–269–5263; E-mail: shenghao.tseng@gmail.com.
NIH Public Access
Author Manuscript
J Biomed Opt. Author manuscript; available in PMC 2009 January 14.
Published in final edited form as:
J Biomed Opt. 2008 ; 13(1): 014016. doi:10.1117/1.2829772.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Amelink et al. developed differential pathlength spectroscopy to investigate local optical
properties of tissue, and this has been successfully applied to study bronchial mucosa.
9
However, this technique requires that all of the chromophores contributing to the measured
signals are known in advance to separate absorption and reduced scattering coefficients from
the measured reflectance. For the case in which constituent chromophores cannot be
determined or the chromophore absorption spectra are not accurate, the empirical mathematical
model will not recover tissue optical properties correctly.
We developed a new fiber-based probe that can be used to determine the optical properties of
superficial in vivo skin using diffuse optical spectroscopy (DOS) in conjunction with a two-
layer diffusion model.
10
This probe employs a highly diffusing Spectralon layer that enables
a diffusion model for small source-detector distances that results in superficial sampling. In
this paper, we briefly review the probe design and the DOS method. To verify the applicability
of this probe for measuring the optical properties of skin, we fabricated two-layer phantoms
with a thin top layer ranging in thickness from 1 to 8mm. The optical properties of the two-
layer phantoms are designed to simulate those of the epidermis+dermis layer and the
subcutaneous fat layer of skin.
8
These layered phantom results illustrate that for samples with
optical properties similar to skin, the superficial diffusing probe of source-detector separation
3mm has an interrogation less than 2mm. Measurements using this probe were subsequently
carried out in vivo at two anatomical locations (volar forearm and palm) on 15 subjects (five
subjects of African descent, five Asians, and five Caucasians). The optical properties obtained
using the superficial diffusing probe correlate closely with the layer structure of skin and
demonstrate similarities to those of ex vivo skin determined from integrating sphere
measurement techniques
6,7
(Caucasian skin μ
a
=0.03 to 0.06/mm and
=1.8 to 2.8/mm at the
wavelength of 650 nm).
2 Materials and Methods
2.1 Layer Structure of Skin
Skin is a layered heterogeneous medium that consists of epidermis, dermis, and subcutaneous
fat, as illustrated in Fig. 1. The epidermis is composed primarily of several cell layers of basal
keratinocytes and ranges from tens to hundreds of micrometer in thickness, depending on
anatomic location. In normal skin, melanocytes within the epidermis are the primary source of
apparent skin color. The dermis, which may be a few hundred micrometer to several millimeters
thick, underlies the epidermis and provides skin with mechanical integrity via a meshwork of
structural proteins including collagen and elastin. A capillary vascular supply permeates the
papillary dermis. Capillary blood flow, structural proteins, and to a lesser extent, cells (such
as fibroblasts) all contribute to the absorption and scattering properties of the dermis layer. For
most anatomic locations, a subcutaneous fat layer, which is composed of adipose, connective
fibrils and blood vessels, underlies the dermis. The scattering and absorption properties of
subcutaneous tissue are introduced by adipocytes, structural fibrils, and blood.
2.2 Instrumentation
A steady state frequency-domain photon migration (SSFDPM) instrument, which was been
described in detail elsewhere
11,12
and successfully applied to deep tissue optical property
studies,
13,14
was employed to carry out measurements in this study. In brief, the instrument
consists of two subsystems: (1) a steady state light source and spectrometer and (2) a bank of
frequency-modulated laser diodes and an avalanche photodiode. The steady state subsystem
includes a tungsten halogen light source (Ocean Optics, Model HL2000) and spectrometer
(BWTek, Model 611) to record wavelength-dependent reflectance from the sample under
investigation. The frequency domain photon migration apparatus employs six laser diodes at
six wavelengths—661, 681, 783, 806, 823, and 850 nm—which are sinusoidally modulated at
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frequencies ranging from 50 to 300 MHz. The diffuse reflectance is detected by an avalanche
photodiode (Hamamatsu, Model C5658). The detected phase delay and the amplitude
demodulation introduced by tissues were fit to an appropriate photon diffusion model to
calculate absorption and reduced scattering coefficients for each laser wavelength. These
optical properties were used to scale the reflectance obtained from the steady state subsystem.
By fitting the reduced scattering coefficient to a scattering power law, the full-spectrum reduced
scattering coefficients in the region from 650 to 1000 nm can be obtained.
15
The absorption
coefficient spectrum can subsequently be calculated for the same wavelength region using a
model of light propagation, such as a photon diffusion model or a Monte Carlo model. A
conventional DOS probe consisting of source and detection fibers is usually utilized along with
a standard diffusion model to recover optical properties of tissue.
12
DOS measurements
performed using a source-detector separation less than 8mm generally do not produce
consistently stable optical property results in tissue since the standard diffusion equation fails
to describe photon transport accurately when the source and detector separation is only a few
scattering lengths.
16
In this study, we employed a superficial diffusing probe, as shown in Fig.
2, which is described in detail in the next section, along with a two-layer diffusion model to
determine optical properties of in vivo human skin.
2.3 Modified Two-Layer Geometry
The superficial diffusing probe with the modified two-layer geometry has been validated as a
quantitative tool for accurately deducing optical properties in homogeneous turbid phantoms.
10
In the phantom study, we demonstrated that the recovered optical properties have a maximal
8% deviation from benchmark values. The superficial diffusing probe contains a high-
scattering, low-absorption layer with known optical properties that is placed on the surface of
the sample under investigation. In practice, the superficial diffusing probe is composed of a
Spectralon slab (LabSphere) and two optical fibers [3M Inc., numerical aperture (NA)=0.37,
core diameter=600 μm], as illustrated in Fig. 3. The Spectralon slab has a thickness of 1.5 mm,
an index of refraction of 1.35, and μ
a
and
of 10
−5
/mm and 50/mm at a wavelength of 660
nm, respectively. Our studies thus far have shown that for measurements on skin, a Spectralon
slab of 1.5 mm thickness provides us with adequate signal and is also thick and rigid enough
to prevent a curved probe surface. The source fiber is mounted flush with the top surface of
Spectralon and the detection fiber penetrates through the Spectralon and is flush with its lower
surface. Source and detection fibers are attached to the Spectralon with epoxy and their
separation is 3mm. In this geometry, all detected photons are constrained to travel through the
Spectralon scattering layer before passing through the sample of interest and arriving at the
detector. The measured reflectance from both frequency-domain and steady state subsystems
is described by a modified two-layer diffusion model. The modified two-layer diffusion model
is an extension of a general two-layer diffusion model proposed by Kienle et al.
17
In the two-
layer model, there are two boundaries: the air/layer-1 (high-scattering layer) boundary and the
layer-l/layer-2 (sample layer) boundary. To apply the model to the superficial diffusing probe,
the source is modeled as a point source pulse located beneath the air/layer-1 boundary at a
distance of , and the detector is located at the layer- l/layer-2 boundary at a distance
from the source. Extrapolated boundary conditions are applied and the fluence rate and z
component of the flux of different layers are continuous at the layer-l/layer-2 boundary.
For the purpose of modeling, the high-scattering layer is considered homogeneous and extends
infinitely in lateral directions, and the sample is assumed homogeneous and semiinfinite in
lateral extent in the two-layer diffusion-based model. The actual diameter of the Spectralon
slab is 10 mm, and we have demonstrated that the modified two-layer diffusion model can be
used to recover the accurate optical properties of semiinfinite homogeneous phantoms.
10
However, because tissues are inhomogeneous, the next step in characterizing this method is to
test the probe and model using two-layer phantoms. These phantoms have top layers of various
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thicknesses as described in the following. This enables us to methodically characterize the
performance of the probe and its applicability for measuring in vivo skin.
3 Validation of the Applicability of the Superficial Diffusing Probe for
Measuring In Vivo Skin Using Two-Layer Phantoms
To investigate the interrogation depth and the accuracy of recovered optical properties of our
probe as it is applied to measure in vivo skin, we designed the optical properties of two-layer
phantoms according to Simpson’s ex vivo measurement results.
6
Five two-layer and two
homogeneous silicone phantoms were made by mixing polydimethylsiloxane (Eager Plastics,
Illinoes), catalyst, titanium dioxide (scatterer), and India ink (absorber) in disposable
polystyrene beakers. The substrates of phantoms were fabricated first by mixing 440 mg of
titanium dioxide, 500 μl of India ink, and 500 ml of polydimethylsiloxane. The substrates were
designed to have optical properties similar to subcutaneous fat.
17
Five “bulk” substrates having
thickness larger than 50 mm were poured at the same time so as to have same optical properties.
One set of “top layer” phantoms, having optical properties that differ from those of the
substrate, were then prepared. We added 440 mg of titanium dioxide and 3ml of India ink into
500 ml polydimethylsiloxane to make the optical properties of the top layer phantoms similar
to those of light skin epidermis-dermis. We designed the optical properties of the top layer
phantoms to mimic those of light skin epidermis-dermis.
17
Once the bulk substrate material
cured, the top layers of different thicknesses were poured and allowed to cure. This resulted
in five two-layer phantoms having different top layer thicknesses, ranging from about 1 to
8mm. The thicknesses of the top layers were well controlled since the polydimethylsiloxane
was in liquid form before cured, and they were punctiliously measured with a digital caliper.
A single bulk substrate was left without a top layer as a homogeneous semiinfinite control. In
addition, a 50-mm-thick phantom of the top layer material was left alone to cure in the absence
of an underlying substrate. The optical properties of bulk substrate and top layer material at a
wavelength of 785 nm are listed in Table 1.
Figure 3 illustrates the recovered μ
a
and
, values versus top layer thickness of the two-layer
phantoms. Each symbol in the plot represents the average of three measurements, and
deviations are all within 0.1%. Optical properties of samples having a 50-mm-thick top layer
are actually results obtained from measurements performed on the homogeneous phantom. We
consider the results from homogeneous phantoms (top layer and substrate) as the benchmark
optical properties.
In Fig. 3 for top layer thicknesses larger than 2mm, the deviation of recovered μ
a
and and
values is within 5% of the benchmark values. As the top layer thickness decreases to 1 mm,
the deviation increases to 28 and 32% relative to the benchmark values for μ
a
, and ,
respectively. These results indicate that the maximum penetration depth of the majority of
detected photons does not exceed 2 mm. Thus, the top layer is virtually semiinfinite and
homogeneous to the diffusing probe when the top layer is thicker than 2 mm. We estimate that
the average interrogation depth of our superficial diffusing probe will be less than 2 mm when
it is applied to in vivo skin, given that these phantoms were designed to mimic the average
properties of epidermis+dermis. In the study carried out by Simpson et al.,
6
the thickness of
the epidermis+dermis of the samples ranged from 1.5 to 2 mm.
As illustrated in Fig. 3, when the two probes are applied to the two-layer phantoms with 1-mm
top layer thicknesses, the recovered optical property values do not fall between the benchmark
optical properties of the top layer and the substrate optical properties. This is a consequence
of the limitations of the diffusion model, which assumes the sample is homogenous and
semiinfinite, whereas the sample is actually layered. This phenomenon was previously reported
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by Farrell et al.
18
Steady state reflectance measurements by this group using a probe having
source-detector separations ranging between 1 and 10 mm resulted in recovered optical
properties for layered tissue samples that were not simply weighted averages of those of
individual layers of tissues.
Our results
19
support their observations when the top layer thickness of our phantoms is
reduced to less than 2 mm, given a probe with source-detector separation of 3 mm. The modified
two-layer diffusion model we employed for our diffusing probe assumes that samples are
semiinfinite and homogeneous. Based on our measurement results, this assumption does not
seem to be valid when the top layer of our phantoms is reduced in thickness to less than 2 mm.
In the next section, we present in vivo measurement results obtained from volar forearm and
palm skin. Based on these results, we conclude that the interrogation region of our probe is
limited to the epidermis and dermis of the two measurement sites. For sites for which the
epidermis+dermis thickness is within the range from 1 to 2 mm, such as the cheek skin of some
subjects, the interrogation depth of the diffusing probe must be decreased to ensure that the
interrogation region is in the epidermis and dermis. Otherwise, the recovered absorption
coefficients will be dependent on the absorption properties of the epidermis, the dermis, and
the subcutaneous layer. One possible way to further reduce the interrogation depth of the
diffusing probe is to shorten the separation between source and detection fibers. The detailed
characterization of the interrogation depth of the diffusing probe is discussed elsewhere.
19
4 Results of In Vivo Skin Measurements
Measurements using the superficial diffusing probe were taken on the volar forearm and the
palm of 15 subjects. The current in vivo measurements were approved by the Institutional
Review Board (IRB) of the University of California, Irvine. Volar forearm skin usually appears
to be more pigmented than the palm of the hand.
20
Because melanin is localized in the
epidermis, we expect to observe differences in absorption properties acquired from these two
anatomical locations when the superficial diffusing probe is employed. Subjects were five
subjects of African descent, five Asians, and five Caucasians. In the process of collecting data,
the probe was gently placed against the skin at each measurement site. Three measurements
were made at each site to assess both random and systematic errors. In every case, the probe
was physically removed from the measurement site and replaced to perform measurement
again. Variations in the recovered optical properties of different measurements at a same site
are within 5%.
The optical properties of volar forearm skin of one subject of African descent, one Asian, and
one Caucasian measured with the superficial diffusing probe are plotted in Fig. 4. Lines and
discrete data points in Fig. 4 represent steady state and frequency-domain recovered optical
properties, respectively. In Fig. 4(a), the absorption spectrum of the subject of African descent
and those of the Asian and the Caucasian subjects have distinct values and trend in the
wavelength range from 650 to 850 nm. The monotonic decrease of the subject of African
descent’s absorption spectrum in the 650 to 850 nm range corresponds to melanin absorption.
21
The absorption features of oxyhemoglobin and deoxyhemoglobin are weakly demonstrated
in the Asian’s and the Caucasian’s absorption spectra, but they are not apparent in the subject
of African descent’s absorption spectrum. This is likely due to the reduced interrogation depth
of the diffusing probe when applied to dark skin relative to depths probed in light skin, and the
fact that the absorption of melanin is much stronger than that of hemoglobin in the 650- to 850-
nm window. The lipid absorption feature at 930 nm cannot be observed for all absorption
spectra. We therefore estimate that the interrogation region of the diffusing probe is limited to
epidermis and dermis when it is applied to volar forearm skin. Figure 4(b) illustrates the reduced
scattering coefficient spectra obtained from the volar forearms of the three subjects. The
reduced scattering coefficient reflects the structure and density of scatterers in the skin such
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as collagen fibrils, cells, and intracellular structures. Reduced scattering coefficients of tissue
in the near IR range usually follow a power law , where λ is the wavelength, and a and
b characterize scatter density and average size, respectively.
22
Similar scatter size and different
density is found in African descent and Asian data, while the Caucasian seems to have different
size and density than other categories. The differences in the reduced scattering coefficient
magnitude between subjects is likely a result of modulation of interrogation depths by the
absorption properties of each skin type. Because the absorption in light skin is relatively small
compared to that in dark skin, the diffusing probe will collect more photons that have passed
through greater volumes of collagen and elastin in the dermis than when applied to dark skin.
Thus, the recovered scattering coefficients of the Asian and the Caucasian are higher than that
of the subject of African descent.
Figure 5 demonstrates the absorption and scattering coefficients from the skin of five subjects
of African descent, five Asians, and five Caucasians. The lines and error bars represent the
average and standard deviation of the three ethnic groups. As shown in Fig. 5(a), the skin of
each ethnic group has a dissimilar slope in the absorption coefficient in the 650-to 850-nm
window, which can be attributed to the different melanin concentration contained in the
epidermis. The superficial diffusing probe is sensitive to the differences in melanin
concentration that exists in the epidermis, and the results demonstrate, for this particular set of
measurements, the ratio of the average absorption coefficients of the subjects African descent’s
skin and the Caucasian skin at the wavelength of 650 nm is about 5. In the 900- to 1000-nm
window, the absorption coefficient demonstrates
12
a water peak at 980 nm. The magnitude of
absorption coefficient of the three groups at 980 nm is very similar (within 2%), indicating that
water concentration of superficial skin of the three ethnic groups is similar, despite the color
of the skin. The average scattering coefficient for the three groups is shown in Fig. 5(b). The
data acquired from the group of African descent still exhibits the lowest scattering magnitude
among the three groups, which is consistent with the trend observed in Fig. 4(b). The average
optical properties of volar forearm obtained using this probe compare favorably to properties
of ex-vivo epidermis+dermis determined by Simpson et al.
6
and Bashkatov et al.
7
using
integrating sphere techniques.
The average optical properties of the heel of the palm measured with a superficial diffusing
probe are plotted in Fig. 6. Surprisingly, in Fig. 6(a), the average absorption coefficients of the
Asian and the Caucasian groups are almost identical in the wavelength window from 650 to
1000 nm. Comparing Fig. 5(a) and Fig. 6(a), we observe that variation in absorption spectra
between subjects is smaller in Fig. 6(a), which is likely a consequence of low melanin
concentration in the palm of the hand regardless of skin color compared to volar forearm.
20
Moreover, the deoxyhemoglobin absorption peak at 760 nm can be discerned for the three
groups. The average reduced scattering coefficient shown in Fig. 6(b) for the three groups is
similar in magnitude and slope, which means that the skin of the palm of the three ethnic groups
have similar composition in terms of average scatterer size and density. Note that the absorption
coefficients of palm skin of the three groups are more tightly clustered than for volar forearm
skin. This suggests that the modulation in the interrogation depth by chromophores in the palm
skin is smaller than that for the volar forearm skin. Hence, the recovered reduced scattering
coefficient of palm skin does not vary as strongly between individuals relative to reduced
scattering coefficient measured in volar forearm skin.
5 Conclusion
we developed a new DOS probe and verified its applicability to determine the optical properties
of in vivo human skin consisting of epidermis+dermis using two-layer phantoms. In the
phantom study, the interrogation depth of our superficial diffusing probe having a 3-mm
source-detector separation was estimated to be less than 2 mm. We applied the probe to measure
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optical properties of in vivo skin of volar forearm and palm for 15 subjects, including five
subjects of African descent, five Asians, and five Caucasians. Our measurement results agree
favorably with those obtained from ex vivo techniques.
6,7
The absorption spectra of the volar
forearm depicted in Fig. 4(a) imply that the probe is sensitive to the melanin that exists in the
epidermis. The absorption spectra of the palm determined using our probe show less difference
between light- and dark-skinned subjects than the absorption spectra measured on the volar
forearm. This is likely a consequence of low melanin concentration in the palm.
20
The
magnitudes of the reduced scattering coefficient obtained for volar forearm skin differ
considerably across the three groups and may be a consequence of the modulation of
interrogation depth of the superficial diffusing probe induced by the skin absorption properties.
The reduced scattering coefficients of the palm of the three skin color groups are very similar,
which suggests that the interrogation regions, the average scatterer size, and the average
scatterer, density of the palm skin of all 15 subjects are similar.
The diffusing probe geometry provides a means for rapidly determining superficial in vivo skin
optical properties over the 650- to 1000-nm range. Such an advance has the potential to impact
light-based diagnostics and therapy. We have begun to work toward the application of this
technology to clinical studies such as skin cancer, oral cancer, and port wine stain therapy. We
are also improving our instrument so that the absorption and scattering properties of tissue at
wavelengths shorter than 650 nm can be acquired, with the hope that access to more prominent
hemoglobin spectral features will enable us to accurately quantify melanin and oxy- and
deoxyhemoglobin concentrations.
Acknowledgments
This work was supported by the National Institutes of Health/National Center for Research Resources (NCRR) under
Grant No. P41-RR01192 (Laser Microbeam and Medical Program: LAMMP), the U.S. Air Force Office of Scientific
Research, the Medical Free-Electron Laser Program (F49620-00-2-0371 and FA9550-04-1-0101), and the Beckman
Foundation.
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Fig. 1.
Layer structure of skin.
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Fig. 2.
Geometry of a superficial diffusing probe.
Tseng et al. Page 10
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Fig. 3.
Optical properties (a) μ
a
and (b)
recovered from two-layer phantoms having top layer
thicknesses from 1 to 8 mm. The dot-dotted and dash-dotted lines represent benchmark optical
properties of the top layers and the substrate of two-layer phantoms, respectively.
Tseng et al. Page 11
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Fig. 4.
Optical properties (a) μ
a
and (b) of in vivo volar forearm skin of one subject of African
descent, an Asian, and a Caucasian measured with a superficial diffusing probe. Discrete data
points are recovered from frequency-domain photon migration (FDPM) measurements and
lines represent broadband optical property spectra of the three subjects.
Tseng et al. Page 12
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Fig. 5.
Broadband optical properties (a) μ
a
and (b)
of in vivo volar forearm skin of five subjects of
African descent (solid line), five Asians (dashed line), and five Caucasians (dotted line)
measured with a superficial diffusing probe. The error bars represent the standard deviation of
optical properties in each group.
Tseng et al. Page 13
J Biomed Opt. Author manuscript; available in PMC 2009 January 14.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Fig. 6.
Broadband optical properties (a) μ
a
and (b)
of in vivo palm skin of five subjects of African
descent (solid line), five Asians (dashed line), and five Caucasians (dotted line) measured with
a superficial diffusing probe. The error bars represent the standard deviation of optical
properties in each group.
Tseng et al. Page 14
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NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Tseng et al. Page 15
Table 1
Optical properties of bulk substrate and bulk top layer materials at a wavelength of 785 nm.
Substrate Top Layer
μ
a
(1/mm)
0.005 0.031
μ’
s
(1/mm)
1.10 2.14
J Biomed Opt. Author manuscript; available in PMC 2009 January 14.
