# In Vitro Assessment of Optical Properties of Blood by  Applying the Extended Huygens-Fresnel Principle to

Authors: Dan P. Popescu; Michael G. Sowa
Journal: International Journal of Biomedical Imaging
 (2008)

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## Abstract

A direct method for the measurement of the optical attenuation coefficient and the scattering anisotropy parameter based on applying the extended Huygens-Fresnel principle to optical coherence tomography images of blood is demonstrated. The images are acquired with a low-power probing beam at the wavelength of 1300 nm. Values of 12.15mm−1 and 0.95 are found for the total attenuation coefficient and the scattering anisotropy factor, respectively. Also, as a preliminary step, the optical refraction index is determined with a precision of two decimal numbers directly from optical coherence images. The total attenuation coefficient and the scattering anisotropy factor are determined with precisions within experimental error margins of 5% and 2%, respectively. Readable OCT signal is obtained for a maximum propagation of light into blood of 0.25 mm. At the maximum probed depth, the measured signal is almost 103 smaller than its initial intensity when entering the sample.


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## Body

## 1. Introduction

Precise measurements of the optical properties of biological
tissues and fluids are  important for a large class of medical diagnostics as
well as therapeutic and surgical approaches that employ light. Often blood is present and therefore its
optical properties need to be considered in the design or deployment of these
optical methods [1–4]. Given its complexity as a propagating environment for
light, a wide array of experimental investigations into the optical properties
of blood has been carried out. There are
methods that directly estimate the light transmission and scattering properties
of blood but they can be applied only to thin blood samples, require
complicated optical setups, and are suitable only for ex-vivo situations
[5, 6]. A number of studies have been
published about blood properties based on widely used indirect methods of
modeling light propagation in turbid environments: wave-scattering theory [7, 8], photon-diffusion theory [1, 9], and inverse Monte-Carlo simulations [10, 11]. Although extensive and rigorous in the mathematical sense, the results
depend on a precise model of the tissue structures as well as the optical
properties associated with these structures.
Often simplified tissue model geometries are employed in these cases to
render analytical or computationally tractable solutions and, when coupled with
imprecise knowledge of the various optical parameters, the accuracy of these
indirect methods can be questioned. In
this paper, we demonstrate a simple and efficient method to directly determine
a number of clinically relevant optical parameters such as the index of
refraction, the attenuation coefficient, and the scattering anisotropy factor
of blood. These parameters are measured at 1300 nm, a promising wavelength to
be used in clinical devices for probing highly scattering biological samples
because it is scattered less than light at shorter wavelengths. The estimations
of the attenuation coefficient and of the anisotropy factor are based on a
numerical algorithm fit on the experimentally acquired optical coherence
tomography (OCT) heterodyne efficiency curve. The connection between these
parameters characterizing blood and the dependence on the propagation distance
within blood of the detected OCT signal is provided by the extended
Huyghens-Fresnel principle [12].
Previously, this optical method has been used successfully in
determining the scattering properties of various biological samples as well as
phantom probes of blood [13–16]. The OCT measurements presented here are
acquired in fresh blood that flows freely within a system of flow cells. The
results obtained in this configuration could be relevant in designing and using
intravascular OCT catheters. Also, the viability of the extended
Huygens-Fresnel principle is verified for OCT probing beams at low powers.
Checking the viability of this principle at low intensities is necessary
because in clinical settings the investigating beam could be strongly quenched
while passing through other tissues before reaching the region of interest.


## 2. Experiment

OCT images are recorded using radiation emitted
from a super-luminescent SLD-571-HP diode with the central wavelength at 1300 nm
and a coherence length of 14μm
(full width at half maximum) measured in air. Due to its
lower absorption and scattering losses in tissues when compared to visible and
shorter near-infrared wavelengths, the spectral region around 1300 nm has the
potential to become important for a number of biophotonics applications [17–19]. In both arms of the interferometer, sample and reference, light is
guided through single-mode fibers terminated with collimating lenses. In the
reference arm, a rapid scanning optical delay line with a constant velocity of
600 mm/s modulates the optical field. A weakly focusing lens with a 48-mm focal
distance and a focal depth of 1.6 mm is located near the exit of the sample
fiber for the purpose of focusing the collimated light onto the sample. The estimated numerical aperture of the
sample arm optical configuration is 0.025. Such a small
value of the numerical aperture together with the proper positioning of the
probed region within the depth of focus provides a beam that could be
approximated as collimated along the axial section of the scanned volume. This
configuration ensures that the beam divergence does not influence the intensity
of the OCT signal along the depth of interest. The fiber-lens assembly is mounted on a computer-controlled horizontal
translation stage that can scan the focused spot of the illuminating beam along
the surface of interest with a maximum spatial resolution of 1 μm.
The power of the beam exiting the sample arm of the interferometer is 2 mW.Fresh porcine blood is acquired from an
abattoir immediately after sacrifice.
Using a Masterflex pump, blood is continuously circulated in a
closed-loop circuit through two consecutive 48/Q/2 Starna flow cells at a
constant flow rate of 1 mL/min. One cell has the thickness of its flowing
channel of 2 mm while the other flow channel in the other cell is 0.2 mm wide.
Blood is flowing normal to the propagation direction of light exiting the
focusing lens that terminates the sample arm of the OCT system. The measured
haematocrit, that is, the ratio of the volume of red blood cells to the entire
volume of blood, stayed constant at 44% for the duration of the experiment. In
order to avoid coagulation, 2 mL/L of heparin was added to the blood immediately
after acquisition. Heparin was added at
a rate of 1 mL/L at 1-hour intervals for the remainder of the protocol. The
blood was kept at room temperature for the duration of the experiment.


## 3. Results and Discussion

The OCT image presented in Figure1(a) is
obtained by acquiring a set of 1600 consecutive depth scans (also known as line
scans or A-scans). This image shows the detected signal backscattered by blood
cells that are flowing through the 0.2-mm cell during its acquisition time. To
eliminate the random thermal, mechanical, and electronic noise, each depth scan
is averaged 10 times. The acquisition time required for such an image is
fifteen minutes. The image presents itself as a speckled pattern that is
generated by the portion of the light flux that reaches the OCT detection
system after it is scattered at the interfaces of blood cells with the
surrounding plasma. This image shows the entire cross-section of the flow
channel crossing through the 0.2-mm cell. In this case, both blood and glass
interfaces are distinguishable and the optical distance between them can be
assessed directly from the OCT image. Knowing the width of the flow channel,
the value of the optical refraction index of blood is readily obtained by
dividing the optical distance between the upper and lower blood/glass
interfaces determined when the channel is filled with blood with the width of
the flow channel. The value obtained for the optical index of refraction for
blood at 1300 nm by this method is nblood=1.39±0.05. The
uncertainty in the measured optical refractive index arises from both the
specified tolerance in the width of the flow cell channel, 0.01 mm (Starna Inc.
catalog), and the imprecision introduced by the finite coherence length of the
OCT source, ~0.014 mm.(a) OCT image of blood flowing
through the 0.2-mm cell. Both blood/glass interfaces are visible. (b) OCT image of
blood flowing through the 2-mm cell. The interrupted white line marks the 600th
A-scan. Both images are composed of 1600 A-scans.
(a)(b)Another two-dimensional OCT image, this time of
blood flowing through the 2-mm flow cell, is presented in
Figure1(b). As light penetrates deeper inside blood, it is reflected, scattered,
and absorbed by various cellular aggregates and fluidic blood components. Due
to the strong light scattering environment that is flowing blood, there is no
recorded OCT signal that comes from the second glass/interface, that is, the
interface located at a 2-mm distance from the interface through which light enters blood. It is known that while light
propagates through blood, it undergoes diffuse reflection from the blood cell membranes and therefore
the backscattered OCT signal is weak. Also, according to
[11, 20], the scattering anisotropy factor for
blood measured at various wavelengths is reported to be between 0.94 and 0.995.
Such values of the anisotropy factor are characteristic to a strong forward
scattering of light in blood and translate into a reduced probability for its
backscattering toward the OCT detection system. The cumulative effect of
multiple scattering events is another factor reducing the detected signal. By
undergoing multiple interactions within the turbid environment, part of the
probing light flux is pushed out from the field of view of the objective lens
or beyond the spatial detection gate imposed by the optical delay line in the
reference arm. These factors, plus the low power of the interrogating beam,
contribute to an OCT signal that is detected after probing a maximum distance
of only 0.25 mm within blood.An example of a demodulated A-scan
interferogram is indicated  in Figure2 by the dotted line. It shows the signal
generated into the OCT detection system through backscattering of light by
blood cells during the 600th A-scan. The position of this particular A-scan is
marked with a white interrupted line in the two-dimensional OCT
image from Figure 1(b). In Figure 2, the specular reflection peak corresponding to the glass/blood
interface is eliminated in order to better emphasize the signal generated only
by the diffuse reflections occurring within the blood environment. The noisy
profile of the single A-scan is common for OCT scans of highly scattering media
and can be attributed to the random positions of scattering centers and to the
speckle noise generated by
multiple scattering.  Speckle noise
occurs in OCT imaging applied to turbid media because of multiple scattered light that experiences
changes in the travel distance relative to its initial ballistic path
reaching the detection
system [17, 21]. In order to accurately estimate the propagation properties of
light in a highly scattering medium, it is necessary to reduce the speckle
noise. In OCT images, speckle noise can
be suppressed by adding spatially independent scans [22–24]. In the presented
case, because of the blood flow, there is a dynamic distribution of cellular
aggregates that act as scattering centers, which in turn induces variations of
the speckle pattern from one A-scan to another. Adding (compounding)
independent individual A-scans with uncorrelated speckle patterns results into
a smoothed depth profile where some of the speckle noise as well as the noise
generated by random electronic and thermal variations in the OCT detection
system are eliminated. Such a compounded profile is exemplified by the
continuous curve from Figure 2, which is obtained by compounding 1000
individual A-scans. To account for the scaling difference between profiles, the
single scan as well as the compounded one is normalized to unity in
Figure 2.Figure 2
Single OCT depth-line scan (dotted line)
compared to the compounded profile (continuous curve). The single-scan line is
the 600th A-scan marked with a white interrupted line in Figure1(b). The
compounded profile results from the summation of 1000 consecutive A-scans.As light penetrates deeper inside blood, the
recorded OCT signal decreases due to both optical absorption and scattering.
Within the depths probed in our experiment, scattering is predominantly
responsible for dissipation of the near-infrared signal while absorption is
responsible for less than 0.5% of the total radiation loss [17]. Therefore, in
the following we will consider that the profiles of individual and compounded
depth scans are exclusively shaped by the scattering properties of blood.The aggregate recorded OCT signal power,〈i2(z)〉,
corresponding to the OCT signal received from a given depth, z , inside
the blood environment can be expressed as the product between the mean square
heterodyne signal in the absence of scattering, 〈i2〉0,
and the heterodyne efficiency, presented as the sum of three terms that account
for the scattering losses [25]:(1)〈i2(z)〉=〈i2〉0×[exp(−2μz)+4exp(−μz)[1−exp(−μz)]1+r2(z)+[1−exp(−μz)]2r2(z)]. In
(1), μ represents the scattering coefficient and r2 is the halo parameter, that is, the ratio between the squares of the 1/e irradiance radii in the z-plane in the presence and absence of
scattering. The halo coefficient is a measure of the lateral coherence length
including its dependence on the penetration distance, that is, the shower
curtain effect [25].As shown by the compounded profile in Figure2, the
light penetration in blood for which a readable OCT signal is obtained for this
particular experimental configuration is approximately 0.25 mm, which is small
when compared to the focal distance (48 mm) of the objective lens used in the
sample arm. Besides the single-backscattered component, there is also
multiple-scattered signal that is recorded in our OCT measurements despite the
low depth of penetration into the turbid environment [26–28]. The following
fact has to be considered in our analysis: blood cells are not point-like
scattering centers but disk-like entities with diameters around 8 μm and thicknesses of approximately 3 μm that occupy a certain volume in space
to the extent that light spends an amount of time propagating through the cell
ensemble comparable to the time spent propagating into the blood plasma.
Therefore, because of intra- and intercell multiple light reflections occurring
at the cell/plasma interfaces, OCT signal that experienced multiple scattering
events is recorded even after short distances (tens of μm) of propagation through blood. The
early onset of a multiple-scattered component in the recorded signal generated
when light propagates in blood phantoms or blood/saline mixtures has been
documented in previous publications [26–28].
Also, the amount of multiple scattered signal increases as the OCT
signal is recorded from deeper within the scattering environment [26–28].In our experimental configuration, the depth within
blood that is probed is centered at the waist of the weakly focused probe beam.
Therefore, the lens-induced axial variation in its intensity has a minimal
impact on the recorded OCT profile. Avoiding the influence of the beam
divergence on the intensity of the compounded profile is helped also by the
small value of the numerical aperture of the optical system from the OCT sample
arm, that is 0.025.Within these experimental conditions and taking into
account the configuration of the OCT system sample arm, the halo factor can be
expressed as(2)r2(z)=1+Cμz3. In (2), C is a constant depending on
sample parameters like the blood refraction index and the scattering anisotropy
parameter, g, as well as on instrumental constants: the effective
numerical aperture, N.A., of the objective lens used to focus the probing beam
onto the sample and the free-space wavelength of the source, λ:(3)C=8π23(N.A.λ)2(1−g)nblood2.The
term contained in the brackets in relation (1), that is, the heterodyne
efficiency which can be also expressed as the 〈i2(z)〉/〈i2〉0 ratio, is fit with the experimentally measured
and normalized compounded OCT signal power shown in Figures 3(a)
and 3(b). Having previously measured the index of refraction of blood and knowing the
wavelength as well as the effective numerical aperture, the latter precisely
measured using the technique described in
[29, 30], only the
scattering coefficient and the anisotropy parameter are used as fitting
parameters. The numerical fit with the experimental heterodyne efficiency curve
is performed up to the point where the signal is 1.5 times higher than the
detection shot noise. Values of 12.15 mm−1 and 0.95 are obtained for
the total attenuation coefficient and for the scattering anisotropy factor.
These values are consistent with the values obtained in [11]. When compared to
results determined at visible and shorter near-infrared wavelengths, the trend
of decreasing scattering coefficients for longer wavelengths in the
near-infrared region is also confirmed.
This trend has been both experimentally observed and theoretically predicted
in several publications [11, 20, 31].Best numerical fit with the
experimental heterodyne profile is obtained for values ofμ=12.15 mm−1 and g=0.95 for the attenuation coefficient and anisotropy
parameter, respectively. The numerical fit is the middle line that follows
closely the experimental heterodyne efficiency curve shown in both (a) and (b)
parts of the figure. (a) Variations of the numerical fit induced by
changes of ±0.7 mm−1 in the attenuation coefficient with the
anisotropy scattering factor kept at g=0.95. (b) Variations in the numerical
fit induced by variations of ±0.02 in the scattering anisotropy parameter with
the attenuation coefficient kept at μ=12.15 mm−1.
(a)(b)The anisotropy factor estimated in this work by
applying the extended Huygens-Fresnel principle to the compounded OCT profile
has a value that is approximately 4% smaller than values theoretically
calculated or measured from transmission-type experimental arrangements. In
such experimental setups, the amount of scattered flux is measured or
calculated at detection points located at distances much greater than the
dimensions of the volume where the scattering takes place. Therefore, the
signatures of individual scattering centers, blood cells in the case under
study, are lost because only the far-field flux is measured. The far-field flux
is a quantity that is an averaged result of the overall scattering events
occurring in the entire probed volume. Meanwhile, due to the interference-based
axial sectioning capabilities and due to the coherence-imposed limitations on
the detected signal, only the scattering properties of blood constituents from
specific locations confined within a small volume are assessed with an
OCT-based configuration. More clearly, in an OCT configuration the detected
signal can be envisioned as portioned into units that are the result of light
interacting only with a small number of cells located within a sample volume
defined by the coherence length of the OCT source and the area of the probing
beam. Therefore, the detected OCT signal is sensitive to the optical and
geometrical characteristics of the individual blood cell and the morphological
discontinuities occurring on microscopic scales can affect its outcome.Figures3(a) and 3(b) help in determining the
sensitivity of the fitting procedure to small variations of the attenuation
coefficient and anisotropy scattering parameter. For comparison, curves
corresponding to attenuation coefficients of 11.35 mm−1 and 12.85 mm−1 are calculated and shown
in Figure 3(a) with the anisotropy parameter kept at
0.95, the value obtained from the numerical fit. Similarly, curves with two
different anisotropy scattering factors, 0.97 and 0.93, are plotted
in Figure 3(b) using the attenuation coefficient numerically obtained from fitting the
compounded OCT profile. Differences between the curves obtained with these
values of the attenuation coefficient and anisotropy factor and the curve that
provides the best numerical fit to the experimental data are obvious from the
figure. The values used to derive the unfit curves are used to estimate the
precision of the procedure. The root-mean-squared error (RMSE) provides a
quantitative measurement of the deviation of the numerical models from the
experimental data. The smallest RMSE is of course obtained for the numerical
fit. As a comparison among the other four cases, the smallest RMSE is obtained
for g=0.97. That RMSE value is still 35% greater than the one corresponding to
the numerical fit.


## 4. Conclusions

A method based on the extended Huygens-Fresnel
principle applied  to time-domain OCT measurements is demonstrated to directly
estimate optical parameters of blood. By
employing this procedure, the use of phantom samples prepared under
predetermined physiological conditions as well as complicated reconstruction
algorithms could be avoided. The research presented here fills a knowledge gap
regarding the optical interaction between blood constituents and light at 1300 nm.
Knowing the refractive index of blood and the attenuation induced in the
signal by light scattering enables a more precise estimation of the effective
propagation distance of OCT signal into blood for a system configuration
similar to an OCT-based intravascular catheter design. The effective depth that
can be probed within blood before the signal becomes imbedded into the shot
noise is a useful parameter to be known when designing and using OCT-based
intravascular catheters. Values of 12.15 mm−1, 1.39, and 0.95 are
estimated for the total attenuation, the refraction index, and the scattering
anisotropy factor, respectively. Both the attenuation coefficient and the
anisotropy factor are determined simultaneously by applying the extended Huygens-Fresnel
principle to the experimental heterodyne efficiency curve. The refraction index
of blood is determined to a second decimal precision while the total
attenuation coefficient and the scattering anisotropy parameter values are
within experimental error margins of 5% and 2%, respectively.



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