Chapter-1 - Methods To Read

Chapter 1. Glucose Diagnostic Methods 1.1.
Introduction
1.1 Introduction
s per the survey and case studies reported by World Health Organization
A (WHO), more than 80% of people around the world suffer from diabetic
metabolic control (WHO, 2010). The data base also indicates that India
is leading the world on largest number of diabetic patients in a single country. Also, it is
the second cause of major number of deaths in the world. Diabetes is a disordered state
of the carbohydrate in the human body which is known as diabetes mellitus (DM) and
is also called a chronic disease. In a diabetic patient lack of insulin causes higher level of
blood glucose concentration. Diabetes is normally classified as type I and type II depend-
ing on the production of insulin. Insulin is a hormone, produced in the langerhans cells
in the pancreas which stimulates the glucose in human body. Insulin dependent Diabetes
Mellitus (IDDM) is called the type I diabetes and accounts for 5% - 10% of all cases which
happens due to the shortage production or lack of insulin from pancreas and normally
occurs before the age of 15 years. Type II diabetes is a Non-insulin dependent Diabetes
Mellitus (NIDDM) caused by the body’s ineffective use of insulin which occurs in 90% -
95% of the diabetic population and mostly occurs in people at middle age. Any kind of
diabetes can be dangerous because long-term excess of glucose (hyperglycemia) can cause
blindness, damaged nerves and kidneys (renal failure), or even increase the risk of heart
disease, strokes, birth defects and other chronic conditions (ADA, 2006). Hyperglycemia
cause damage to the nervous and vascular systems. Furthermore, in critical patients hy-
perglycemia increase the mortality with up to 40 %. Low levels (hypoglycemia), however,
coma and even death (Amaral and Wolf, 2008; Garg et al., 2006; Larin et al., 2003; WHO,
2010).
Significant efforts have been made by several scientific groups and industries in the past
few decades to develop a device for the measurement of blood glucose adopting invasive,
minimally-invasive and non-invasive methods. The biochemical analysis of the glucose
monitoring gives the more accepted method for diabetes diagnosis. Blood fluid is a
combination of cells and plasma or serum. From the last few decades, several methods
have been used in clinic for the quantitative estimation of glucose in blood either in whole
blood, plasma or serum. In the biochemical analysis, blood glucose is known as a blood
sugar. Conventional available techniques require few µl amount of blood from the human
for the measurement of blood glucose and is painful. The variations in testing under
biochemical methods are (Larin et al., 2003)
• Fasting Blood Sugar (FBS) : The blood sample is collected after the patient fasts
for 12 hours or overnight.
• Post-Prandial Blood Sugar (PPBS) : After the patient fasts for 12 hours, a meal is

Jitendra Solanki 10 D.A.V.V., Indore
Chapter 1. Glucose Diagnostic Methods 1.2. Chemical Methods
given which contains starch and sugar (≈ 100 gm). Blood is collected 2 hours after
the ingestion of the meal.
• Random Sample : Blood is collected any time without prior prepration of the
patient.
Complications and mortality in diabetic and critically ill patients may be controlled
using the continuous blood glucose monitoring and tight glucose control. Every diabetic
patient should get continuous, painless blood glucose monitoring and facilitate tight blood
glucose control using noninvasive techniques. Therefore, the development of noninvasive
continuous blood glucose monitoring is of vital importance.
A biosensor for noninvasive blood glucose analysis can be based on different optical ap-
proaches. These approaches include optical coherence tomography (OCT), near-infrared
(NIR) absorption and scattering, polarimetry, Raman spectroscopy, photoacoustic, thermo-
optical studies on human skin and fluorescence. Despite significant efforts, the described
techniques for noninvasive monitoring of glucose concentration have faced limitations as-
sociated with low sensitivity, accuracy and insufficient specificity of glucose concentration
measurement at physiological range (4-30 mM or 72-540 mg/dl). A noninvasive glucose
sensor should have at least the same or better accuracy and have an acceptable speci-
ficity (Amir et al., 2007; Larin et al., 2003). Although, these techniques are promising
and required further development to provide clinically acceptable, accuracy, specificity
and reproducibility. Optical coherence tomography (OCT) was proposed previously for
blood glucose monitoring and was tested in- vivo and in-vitro. It was shown that OCT
is capable of detecting changes in blood glucose concentrations as small as a clinically
acceptable value of 20 mg/dl (Amir et al., 2007; Kuranov et al., 2007; Larin et al., 2003).
1.2 Chemical Methods
1.2.1 Invasive Method
Pathologist measures the level of the blood glucose in human subject using invasive
method which provide the most accurate measurements and are comparatively inexpen-
sive. It requires small volume of blood-sample 3-6 µl using test strips or biosensor and
blood collection is nearly painless (Bazaev and Selishchev, 2007; Tura et al., 2007). Glu-
cose Oxidase Detection (GOD) is used in pathology laboratory for the measurement of
the blood glucose in human subjects. Glucose oxidation reaction catalyzed by glucose
oxidase (GOD) and is defined as
Glucose (C6 H12 O6 ) + O2 + H2 O −

GOD
−−→ Gluconic acid + H2 O2 (1.1)

Glucose oxidase catalyses the oxidation of glucose to D-gluconic acid and hydrogen per-
oxide. This (H2 O2 ) is broken down to water and oxygen by a peroxidase in the presence
of an oxygen acceptor which itself is converted to a coloured compound, the amount of
which can be measured using colorimetrically (Varley, 1969; Wilson et al., 1992). It is
CH2OH
CH2OH
HO O O
OH OH
OH acyclic - D - glucose OH
OH
OH HCO
OH OH
alpha - D - glucofuranose alpha - D - glucopyranose
OH
HO
OH
beta - D - glucofuranose beta - D - glucopyranose
OH
CH2OH CH2OH
O
HO CH2OH OH
O OH
OH
OH
OH
OH OH
OH
Figure 1.1: Anomers of D-glucose in an aqueous solution (Poddar et al., 2008).
highly specific for β-D-glucose and does not act on α-D-glucose. It is used in the de-
termination of free glucose in body fluids. Although, specific for β-D-glucose, glucose
oxidase can be used to measure the total amount of glucose. This is because, following
the consumption of β-glucose, α-glucose at equilibrium is converted to the β-form by mu-
tarotation is shown in Figure 1.1. The consumed oxygen or the ensuring production of
gluconic acid or hydrogen peroxide (H2 O2 ) is in direct proportion to the glucose content.
The glucose oxidase method is characterized by high sensitivity, accuracy and reliability.
Traditional electrochemical methods such as potentiometry can be used to determine the
glucose content during oxidation reaction. This method is used in various auto-analyzers.
However, invasive monitors are inappropriate for continuous glucose monitoring (Kuranov
et al., 2007; Tura et al., 2007; Varley, 1969).
1.2.2 Minimally Invasive Method
Minimally invasive methods also require tissue fluid and induces minimal skin injury in
the subject. These techniques are not suitable but are reliable for the estimation of blood
glucose. Virtually all of the techniques for blood glucose monitoring have a common
method of measurement. In every case an energy source, for example, light or electric
current, is used to investigate a sample is shown in Fig. 1.2. The sample may be a fluid
directively in situ or extracted, or a reporter molecule may be used, again either in situ

Figure 1.2: A schematic for the measurement of the glucose level using minimally invasive
method (Ginsberg, 1992).
or in extracted fluids. The energy is modulated by its interaction with the sample and
the alteration is analyzed to determine the glucose value of the sample.
A wide variety of fluids can be measured: blood, interstitial fluid, aqueous humor, lymph,
urine, sweat, saliva, tears, cerebrospinal fluid and others (Ginsberg, 1992).
1.2.3 Continuous Glucose Monitoring (CGM)
CGM provides additional temporal information, such as trends, magnitude, duration

and frequency of glucose level fluctuations. This information can aid in the identification
and prevention of unwanted hypo- and hypoglycemic episodes (e.g., during sleep). Fur-
thermore, it can activate alarm signals for extreme glucose levels, decrease the nursing
workload in tight glycemic control and facilitate in automatic feedback-controlled insulin
delivery systems, such as an artificial pancreas. CGM can also help adjust therapy, quan-
tify the response in diabetic therapy trials and monitor conditions where tight control
without hypoglycemia is sought (Amir et al., 2007).
1.2.4 Self-Monitoring of Blood Glucose (SMBG)
SMBG levels is highly recommended by the American Diabetes Association as an integral

part of the management strategy for diabetic patients using multiple insulin injections.
SMBG is helpful in achieving glycemic goals as well for patients using less frequent insulin
injections, oral agents, or medical nutrition therapy alone. Nevertheless, because most

Chapter 1. Glucose Diagnostic Methods 1.3. Physical Methods (Noninvasive)
patients use conventional glucometers, the number of daily measurements is limited by

patient compliance and by the price of the disposable glucometer sticks. Consequently,
only intermittent and incomplete profiles of blood glucose fluctuations are generated. A
truly noninvasive glucose-sensing device could revolutionalize diabetes treatment by lead-
ing to improved compliance with recommended glucose levels, thus obtaining hemoglobin
A1c at normal or near-normal levels without increasing the incidence of hypoglycemia
due to intensive insulin therapy. Great advances have been made in glucose measurement
techniques since the first chemical test were introduced in the early 19th century (Boehm
et al., 2009; Kvist and Jensen, 2007).
1.3 Physical Methods (Noninvasive)
Noninvasive determination of glucose can be classified in two broad categories as meth-

ods tracking a molecular property of glucose, or methods tracking the effect of glucose on
tissue and blood properties. The first category depends on tracking an intrinsic molec-
ular property of glucose such as near-infrared (NIR) absorption coefficient, mid-infrared
absorption coefficient, optical rotation, Raman shifts and photoacoustic (PA). These
methods assume the ability to detect glucose in tissue or blood independent of other
body components and also independent of the body’s physiological state. The second
set of methods depends on measuring the effect of glucose on the optical properties of
tissue. These properties include light scattering coefficient of tissue, refractive index of
interstitial fluid (ISF) and sound propagation in tissue.
Various noninvasive optical methods have been developed for the measurement of the
blood glucose in the human subjects by Bazaev and Selishchev (2007); Srivastava et al.
(2013).
1.3.1 Near Infrared Spectroscopy (NIR)
A beam of light spectrum 750 to 2500 nm is focused on the body in the near infrared
spectroscopy. NIR spectroscopy allows glucose measurement in tissues in the range of 1-
100 mm of depth, with a decrease in penetration depth for increasing wavelength values.
The light focused on the body is partially absorbed and scattered, due to its interac-
tion with the chemical components within the tissue. Attenuation of light in tissue is
described, according to light transport theory, by the equation I = Io e−µef f d where I, Io ,
µef f and d are the reflected light intensity, incident light intensity, effective attenuation
coefficient and optical path length in tissue, respectively. µef f = f (µa , µs ), where µa and
µs are absorption and scattering coefficient, respectively. It is well known that chang-
ing the glucose concentration can influence µa of a tissue through changes of absorption

corresponding to water displacements or changes in its intrinsic absorption. Changes in

glucose concentration also affect the intensity of light scattered by the tissue, i.e. µs .
This coefficient is a function of the density of scattering centers in the tissue observation
volume, the mean diameter of the scattering centers, their refractive index and the re-
fractive index of the surrounding fluid. For the case of cutaneous tissue, connecting tissue
fibers are the scattering centers. Erythrocytes are the scattering centers for the blood.
In this method, glucose concentration could be estimated by variations of light intensity
both transmitted through a glucose containing tissue and reflected by the tissue itself.
Transmission or reflactance (localized or diffuse) of the light can be measured by proper
detectors (Amir et al., 2007).
The purpose behind this techniques is that absorption coefficient of glucose in the NIR
band is low and is much smaller than that of water by virtue of the large disparity in
their respective concentrations. Thus, in the NIR the weak glucose spectral bands only
overlap with the stronger bands of water, but also of hemoglobin, protein and fats. As
regards the scattering coefficient, the effect of a medium is non-specific and hence it is
common to other soluble analytes. Another confounding factor is due to the fact that NIR
measurements often reflect glucose concentration in different body compartment, that is,
not only blood but also the interstitial fluid in different body tissues can contribute to the
measured signal. NIR light transmission or reflectance has been studied through an ear
lobe, finger web and finger cuticle, skin of the forearm, lip mucosa, oral mucosa, tongue,
nasal septum, cheek and arm. NIR diffuse reflectance measurements performed on the
finger showed a correlation with blood glucose but predictions were often not sufficiently
accurate to be clinically acceptable. Diffuse reflectance studies of the inner lip also showed
good correlation between blood glucose and indicated time lag which is of few minutes
between blood glucose and the measured signal (Marbach et al., 1993). Salivary glucose
levels (a component of lip measurements) did not reflect blood glucose levels. These
locations were selected for some advantages, such as high vascularization, little fatty
tissue, homogeneous composition and limited temperature variations. It must be noted
that, depending on the considered site, some specific intervals in the NIR band have
been considered and the choice on one specific site also influenced the type of studied
light, i.e. transmitted, or reflected (localized or diffuse) or both. Definitions of NIR
band are not always in agreement : some authors define NIR in the 750-2,500 nm band,
others in 1000-2500 nm band, whereas they define the 700-1,000 nm band as the infrared
band. However, wavelength under 1,000 nm are rarely used for glucose measurements:
some studies have been performed, but without any notable results. Since the major
contribution to the glucose information present in NIR reflectance spectra can be expected
to be from the dermal layer (from glucose present in the ISF and the capillaries), Tura
et al. (2007) have been studied the behavior of dermal ISF with changes in blood glucose.

The selection of the measuring site of human tissue that provides sufficient information
about blood glucose is also important. When a NIR spectrum of skin tissue is measured,
the existence of the stratum corneum is a major problem. To avoid the interference from
the stratum corneum, Heise and Fraser (1997) measured NIR spectra of oral mucosa
using reflection optics based on an ellipsoidal mirror because the oral mucosa does not
contain stratum corneum. The turbidity of most fibrous tissues in visible and NIR (near
infrared) spectral ranges is due to their high scattering with low absorption. The NIR
spectral range encompasses combinations and overtone bands that are broad and weak
(Thennadill et al., 2001).
1.3.2 Mid-Infrared Spectroscopy (Mid-IR)
Mid-infrared (Mid-IR) spectroscopy is based on light in the 2,500-10,000 nm spectrum

(Khalil, 2004). The physical principle is similar to that of NIR. When compared to NIR,
however, due to the higher wavelengths, Mid-IR exhibits decreased scattering phenomena
and increased absorption. For this reason, the tissue penetration of light can reach a few
µm (Brancaleon et al., 2001). In the case of human skin, that corresponds to the stratum
corneum. As a consequence only reflected and scattered light can be considered: there is
no light transmitted through a body segment. On the other hand, a possible advantage
of Mid-IR compared to NIR is that the Mid-IR bands produced by glucose, as well as
other compounds, are sharper than those of NIR, which are often broad and weak. One
strong limitation is the poor penetration. Furthermore, Mid-IR is affected by similar
problems and confounding factors than NIR, despite glucose bands potentially improved.
For instance, some studies have shown significant dependence of skin Mid-IR spectrum
on its water content. Mid-IR is less studied technique compared to NIR for glucose
measurement, probably due to the strong limitation in penetration. Studies are reported
related to finger skin and oral mucosa. Considering it in the 25,00-25,000 nm range, some
authors also provide a broader definition of the Mid-IR band than that reported above
(Tura et al., 2007).
1.3.3 Temperature-Modulated Localized Reflectance
This is again in the field of light scatter-based technique. It is based on the observation
that temperature changes causes variations in the tissues refractive index (which influ-
ences the light scattering), but on the other hand the entity of these changes depend
upon glucose concentration. More, specifically, the temperature modulation of the local-
ized reflected light due to scattering is analyzed. Glucose concentration is estimated with
localized reflectance signals at 590 and 935 nm. In some studies, a probe was placed in

contact with the skin and the probe temperature was varied between 22o C and 38o C. Af-
ter each variation, skin was equilibrated for some minutes. During each of those intervals
some light packets were collected and related to glucose concentration (Yeh et al., 2003).
Several parameters can affect this kind of measurements, both physiological and techni-
cal (such as the probe position). Also a peculiar health status, such as an inflammatory
state with possible fever condition, can affect the measurement. Measurements are usu-
ally performed on the skin of the forearm. In particular, glucose in the dermis layer is
estimated (Kinchiku et al., 2012).
1.3.4 Raman Spectroscopy
The Raman spectroscopy is based on the use of a laser light to induce oscillations and
rotations in molecules of one solution. The consequent emission of scattered light is in-
fluenced by the molecules vibration, which depend on the concentration of the solutes in
the solution. Therefore, it is possible to derive an estimation of glucose concentration in
human fluids where glucose is present. The Raman spectrum usually considered is in the
interval 200-1800 cm−1 . In this band, Raman spectrum of glucose is quite clearly differ-
entiable from that of other compounds. In fact, Raman spectroscopy usually provides
sharper and less overlapped spectra compared, for instance to NIR. Other advantages
are the modest interference from luminescence and fluorescence phenomena. Fixed wave-
length lasers at relatively low cost can be used. Recently, an improvement in traditional
Raman spectroscopy has been proposed (surface-enhanced Raman spectroscopy), which
may increase the sensitivity of the acquisition and/or decreasing the acquisition time
(Hanlon et al., 2000; Yeh et al., 2003).
Main limitations are related to instability of the laser wavelength and intensity and long
spectral acquisition times. Moreover, similarly to other techniques described before, the
problem of the interference related to other compounds remains. The eye is the most
common site. The laser light is passed tangentially through the front of the eye. Another
possible site is human skin, although more confounding compounds, such as lipids, are
present (Caspers et al., 2003; Owyoung and Jones, 1997; Steffes, 1999; Yonzon et al.,
2004).
1.3.5 Polarization Changes
It is based on the phenomenon that occurs when polarized light transverses a solution
containing optically active solutes (such as chiral molecules): the light, in fact, rotates
its polarization plane by a certain angle, which is related to the concentration of the
optically active solutes. Glucose is a chiral molecule and its light rotation properties

have been known for a long time. Indeed, investigation of the polarization changes
induced by glucose is reported to be the first proposed non-invasive technique for glucose
measurement in humans. One advantage of this technique is that it can make use of
visible light, easily available. Moreover, the optical components can be easily miniaturized
(Khalil, 2004).
This technique is sensitive to the scattering properties of the investigated tissue, since
scattering depolarizes the light. As a consequence, skin cannot be investigated by po-
larimetry, since it shows high scattering due in particular to the stratum corneum. More-
over, the specificity of this technique is poor, since several optically active compounds
are present in human fluids containing glucose, such as ascorbate and albumin. However,
specificity can be partially improved by using multiple light wavelengths. Other general
sources of errors are variations in temperature and pH of the solution. The preferential
site for this technique is the eye and, more specifically, the aqueous humor beneath the
cornea. Cornea has in fact low scattering properties, since it does not have the stratum
corneum. However, some sources of errors are due to eye movements and corneal rota-
tions. The corneal birefringence, due to its collagen structure, is another error source.
Moreover, a time delay between glucose concentration in aqueous humor and blood has
been observed and hence it has to be taken into account (Cameron et al., 2001; Rabi-
novitch et al., 1982; Rawer et al., 2002).
1.3.6 Ultrasound and Photoacoustic Techniques
The most used technology based on ultrasound is the photoacoustic spectroscopy, which
is based on the use of a laser light for the excitation of a fluid and consequent acoustic
response. The fluid is excited by a short laser pulse (from pico to nanoseconds), with a
wavelength that is absorbed by a particular molecular species in the fluid. Light absorp-
tion causes microscopic localized heating in the medium, which generates an ultrasound
pressure wave that is detectable by a microphone. In clear media (that is, optically thin),
the photoacoustic signal is a function of the laser light energy in which the light signal is
relatively unaffected by scattering. The photoacoustic spectrum as a function of the laser
light wavelength mimics the absorption spectrum in clear media. However, this technique
can provide higher sensitivity than traditional spectroscopy in the determination of glu-
cose. This is also due to the relatively poor photoacoustic response of water, which makes
easier the determination of compounds, such as hydrocarbons and glucose. The laser light
wavelengths that can be used vary in a wide interval (from Ultraviolet to NIR) (Shen
et al., 2000; Tura et al., 2007). One variation in photoacoustic spectroscopy is based on
combining it with ultrasounds emission and detection. A possible approach is the use
of an ultrasound transducer to locate a bolus of blood in a vessel and then illuminate it

with the laser pulse at a glucose absorption wavelength. The ultrasound transducer also
detects the generated photoacoustic signal. Another approach is detecting ultrasound
signal reflected from a blood vessel before and after photoacoustic excitation. Glucose is
then determined from the difference in reflected ultrasound intensity. A third approach is
exciting a blood bolus via photoacoustic effect: the change in the dimensions and speed
of the excited bolus causes a Doppler shift in an ultrasound directed towards the blood
vessel; glucose is determined from the magnitude and the delay of the Doppler-shifted ul-
trasound peak. The technique is sensitive to chemical interferences from some biological
compounds and to physical interferences from temperature and pressure changes. More-
over, when the laser light transverses a dense media, its contribution to the photoacoustic
signal is due not only to the absorption coefficient but also to the scattering coefficient,
thus possibly resulting in a confounding factor if not taken into account. Indeed, the
photoacoustic spectrum of a dense media is similar to the diffuse reflectance spectrum
rather than the absorption spectrum. On the other hand, the scattering can enhance
the signal and hence it can provide an advantage if properly considered. A technological
disadvantage is that the instrumentation is still custom made, expensive and sensitive to
environmental parameters (Park et al., 2009). A possible body site for measurement is the
eye and, especially, the eye sclera. Other sites are fingers and forearm, with contribution
in glucose determination by blood vessels, by skin and tissues or both.
1.3.7 Fluorescence Technology
This technique is based on the generation of fluorescence by human tissues when excited
by lights at specific frequencies. In the case of glucose, one study demonstrated that
when a glucose solution is excited by an ultraviolet laser light at 308 nm, fluorescence
can be detected at 340, 380, 400 nm, with maximum at 380 nm (Khalil, 2004). It was
also proved that fluorescence intensity was dependent upon glucose concentration in the
solution. Also light in the visible spectrum can be used, but this is more adequate for
studying fluorescence of tissues rather than that of solutions.
In tissues, the use of ultraviolet light could lead to strong scattering phenomena, in ad-
dition to fluorescence. Moreover, even when using different wavelengths, the fluorescence
phenomenon can depend not only on glucose, but on several parameters, such as skin
pigmentation, redness and epidermal thickness. In humans, estimations of glucose con-
centration have been attempted on skin with patented blue light fluorescence technology
(Cameron et al., 2001; Sandb-Moller et al., 2003).

1.3.8 Thermal Spectroscopy
This technology does in fact include different approaches. In thermal gradient spec-
troscopy, it is considered the absorptive effects of glucose on the body naturally emitted
infrared (IR) radiation. Such IR absorption effect of glucose is obviously related to its
concentration. In some studies, the skin was cooled to approximately 10◦ C to suppress
its absorptive effects.
Every variation of body or tissue temperature is a strong confounding effect. It must be
noticed that several factors can induce temperature variations, both physiological (such
as the circadian periodicity) and pathological (for instance, a fever status). A traditional
site for this technique is the skin of the forearm or the finger. However, a different possible
site is the ear: a sensor can in fact be inserted in the ear canal to measure the IR radiation
emitted by the tympanic membrane (Yeh et al., 2003).
1.3.9 Ocular Spectroscopy
This technique is based on the use of a special contact lens where a hydrogel has been
added. A hydrogel wafer with 7 mm of thickness based on boronic acid derivatives was
bonded to the lens. The boronic acid derivative is able to create reversible covalent
bonds with glucose and the phenomenon is influenced by the glucose concentration in
tears. When the lens is illuminated by a light source (such as a laser light), the reflected
light changes its wavelength (i.e. its colour) depending on the entity of the binding
phenomenon, which is related to tear glucose concentration. The light colour changes
can be detected by a spectrometer.
There is a delay between glucose in blood and in tears. Moreover, the use of contact lens,
though it can be considered a non-invasive approach, may be uncomfortable to some
subjects. The measurement site is the eye (Chowdhury et al., 2013; Domschke et al.,
2006; Tura et al., 2007).
1.3.10 Bio-impedance Spectroscopy
The impedance of one tissue can be measured by a current flow of known intensity through
it. If the experiment is repeated with alternating currents at different wavelengths, the
impedance (dielectric) spectrum is determined. The dielectric spectrum is measured in
the frequency range of 100 Hz to 100 MHz. Variations in plasma glucose concentration
induce in red blood cells, a decrease in sodium ion concentration and an increase in potas-
sium ion concentration. These variations cause changes in the red blood cells membrane
potential, which can be estimated by determining the permittivity and conductivity of

the cell membrane through the dielectric spectrum (Ermolina et al., 2000; Hillier et al.,
1999).
Some problems remain to be clarified, such as the effect of body water content and
of dehydration. Moreover, some diseases affecting the cell membranes can also have an
influence that needs to be evaluated. The most known study was performed with a watch
like device, positioned on the wrist (Caduff et al., 2003; Polevaya et al., 1999).
1.3.11 Electromagnetic Sensing
Similar to impedance spectroscopy, this technique detects the dielectric parameters of

blood. However, in the former an electric current is used, while in the latter the electro-
magnetic coupling between two inductors is exploited. The inductors are turned around
the medium under study: in-vitro context, the system can be described by the sketch.
The tubes, simulating the human veins, contain blood. To make the experiment more re-
alistic, the tubes were covered by gelatine, which simulates the tissues surrounding blood
vessels in-vivo context. The electromagnetic coupling of the two inductors is modified
by variations in the dielectric parameters of the solution, i.e. blood in this case. On the
other hand, dielectric parameters of the solution are influenced by glucose and hence an
estimate of glucose concentration can be derived (Tura et al., 2007).
The temperature has a strong effect on the measurement; since it also influences the
optimal investigation frequency, temperature measurement has to be performed and the
information properly considered. Furthermore, the blood dielectric parameters depend
on several components other than glucose (Gourzi et al., 2005, 2003).
1.3.12 Fluid Harvesting Technique
This technique is based on the use of a laser light or ultrasounds to create an array of
microscopic holes (micropores) on human skin. The interstitial fluid, containing glucose,
tends to migrate through the micropores to a glucose sensor (of traditional type) placed
in contact with the skin where the micropores have been created and hence direct glucose
measurement becomes possible. The technique based on laser light is called by some au-
thors biophotonic technique, whereas that based on ultrasound is also called sonophoresis
(Gebhart et al., 2003; Guo et al., 2012).
No limitations or confounding factors have been explicitly described, except that the pos-
sible mismatch between glucose in the interstitial fluid and glucose in blood. The site for
measurement is human skin in general. The arm is probably the most common applica-
tion site. Due to micropore generation, the technique is considered by some authors as

minimally invasive (Mitragotri et al., 2000).
1.3.13 Iontophoresis
Iontophoresis (or, more exactly, reverse iontophoresis) is based on the flow of a low
electrical current through the skin, between an anode and cathode positioned on the skin
surface. An electric potential is applied between the anode and cathode, thus causing
the migration of sodium and chloride ions from beneath the skin towards the cathode
and anode, respectively. In particular, it is sodium ion migration that mainly generates
the current. Uncharged molecules, such as glucose, are carried along with the ions by
convective flow (electroosmosis). This flow causes interstitial glucose to be transported
across the skin, thus being collected at the cathode where a traditional glucose sensor
is placed to get direct glucose concentration measurement. Over the typical range of
iontophoretic current densities (< 0.5 mA/cm2 ), glucose extraction is approximately in
linear relation with the density and duration of iontophoretic current (Kurnik et al.,
1999).
The main drawback of this technique is that it tends to cause skin irritation. This problem
may be avoided by limiting the time interval of the electrical potential application. On
the other hand, a minimum duration is required to get a sufficient amount of glucose
for measurement. Furthermore, this approach cannot be used if the subject is sweating
significantly. There is also discussion as to whether this technology can be able to detect
rapid changes in blood glucose. This is, actually, a general problem of any measurement of
glucose not in blood. The measurement can be obtained with a watch-like device placed
on the wrist. Similarly to the fluid harvesting technique, also iontophoresis is considered
by some authors as minimally invasive. In fact, the glucose is again extracted from the
skin and directly measured. The two techniques are sometimes defined as transdermal
(Pitzer et al., 2001; Tura et al., 2007).
1.3.14 GlucoWatch
GlucoWatch has a wrist-watch format. It measures glucose through the skin using a
disposable pad, which clips into the back of the meter. The pad uses an adhesive to stick
to the skin allowing it to come in contact with a small electrical current, which causes the
reverse iontophoresis. The electrical charges bring glucose to the skin surface where an
enzyme reaction similar to that found in a standard meter occurs. Thus, glucose levels
in the interstitial fluid can be estimated. Compared with finger-stick readings, the meter
measurements have a 15-min lag time. The meter is intended for use to supplement, but
not to replace, information obtained from a standard blood glucose meter. The meter has

23 hour warm-up period. Afterwards, it measures glucose every 10 min (at least in the
latest version, called GlucoWatch G2 Biographer): 3 min of electrical stimulation, then
7 min of glucose measurement. An alarm also occurs when a rapid change is seen in the
blood sugar (more than a 35%) change from any reading in the last hour), when you are
sweating and for any measurements above or below the patient’s target levels. A trend
indicator appears to show the direction of the blood sugar when the current measurement
is more than 18 mg/dl (1 mM/l) higher or lower than the previous measurements. Event
markers can be recorded for activities like meals, insulin intake and exercise (Tura et al.,
2007).
GlucoWatch is reported to possibly have some drawbacks: in fact, it sometimes causes
skin irritation and it may be uncomfortable to use in the daily life of the patients (it
may be inaccurate in the case of changes in body or environmental temperature, in the
presence of relevant movement, or if the patient is sweating). Sensitivity to those factors,
often rapidly changing in the ordinary daily life, may be a limitation also for several other
devices, especially for those designed to be wearable (Garg et al., 2006; Pitzer et al., 2001).
1.3.15 Optical Coherence Tomography (OCT)
Optical coherence tomography (OCT) is based on Michelson interferometer which use

superluminescent diode (SLD) as low coherence light source, a reference arm with moving
stage and a sample arm and a photodetector is used to measure the interferometric
signal (Huang and et al., 1991; Larin et al., 2002). Light backscattered from tissues
is combined with light returned from the reference arm of the interferometer and the
resulting interferometric signal is detected by the photodetector.
Moving the mirror in the reference arm of the interferometer allows scanning the tissues
up to a depth of about 1 mm. By moving the mirror into the sample arm scanning
of the tissue surface is obtained. Therefore, this techniques has unique capability of in
depth and lateral scanning to obtain 2D images with high resolution. Tissue scattering
properties are highly dependent on the ratio of the refractive index of scattering centers
(cellular components, protein, etc.) to the refracttive index of the interstitial fluid. An
increase of glucose concentration in the interstitial fluid causes an increase in its refractive
index, thus determining a decrease in refractive index mismatch and hence of the scatter-
ing coefficient. OCT technique can be sensitive to motion artifacts. Moreover, although
little changes in skin temperature have negligible effects, changes of several degrees have
a significant influence on the signal (Larin et al., 2003, 2002). The body site for measure-
ment is the skin, typically in the forearm. More specifically, glucose concentration in the
interstitial fluid of the upper dermis of the skin is investigated.
Optical coherence tomography (OCT) is a new optical approach to the clinically first

proposed by (Huang and et al., 1991) to noninvasive monitoring of blood glucose con-
centration because of its capability of probing optical properties at different depth in
biological tissue with high resolution. Blood glucose monitoring using standard invasive
and minimally invasive techniques are not approaching to the accuracy but the accuracy
may be provide using OCT. The reliability of optical coherence tomography (OCT) tech-
nique at clinical is increasing due to noninvasive blood glucose monitoring, accuracy and
reproducibility (Huang and et al., 1991).
OCT is a new emerging biomedical novel technology for the quantitative estimation of
blood glucose in human subjects. It integrates a wide range of disciplines, including
fiber optics, interferometry, biomedical imaging, in-vitro and in-vivo studies and clinical
medicine. OCT analogous to Ultrasound: It measures the intensity of back-reflected near-
infrared light to measure the thickness of different biological tissues, which is analogous to
B-mode ultrasound imaging , except that it uses light instead of sound. Sound waves are
easily transmitted in most of the biological tissues and the deep structures in the body can
be imaged using an ultrasound techniques. Ultrasound imaging depends on the reflection
of sound waves and the resolution of its measurement depends on the frequency of the
sound waves. The spatial resolution of approximately 150 µm can be obtained using a
typical ultrasound system. OCT uses light, which provides a significantly higher spatial
resolution than that of any ultrasound technique. OCT images can have axial resolution
of 10 µm, which is 10-20 times greater than standard B-mode ultrasound imaging. Index
matching technology has been used to allow better visualization through blood (Larin
et al., 2002). The OCT technique has several advantages over other optical methods
like high resolution (< 10µm), layer-specific probing capabilities and high dynamic range
(> 100dB) (Fercher et al., 2003; Fujimoto, 2003; Larin et al., 2003, 2002; Poddar et al.,
2008; Podoleanu, 2005; Schmitt, 1999).
1.3.16 Construction of OCT
Optical Coherence Tomography is divided in two types on the basis of its construction as
fiber-optic based OCT and free space based OCT. Time Domain OCT is mostly used for
the monitoring of the blood glucose non-invasively in human subjects. Depth resolved
information in optical coherence tomography (OCT) is obtained using different scanning,
acquisition and processing techniques. OCT is based on the well established white light
interferometry. The quest for higher resolution and faster acquisition of in-vivo images
has ensured OCT a rapid evolution in the last decade (Podoleanu, 2005).
Optical-coherence tomography (OCT) is a new emerging non-invasive and high-resolution
imaging modality which employs nonionizing optical radiation. OCT is derieved from
low-coherence interferometry. OCT is an absolute measurement technique developed

for high-resolution ranging and characterization of optoelectronic components. The first

application of low-coherence interferometry in the biomedical optics field was for the mea-
surement of eye length. Adding lateral scanning to a low-coherence interferometer, allows
depth resolved acquisition of 3D information from the volume of biological material. This
technique has potential to achieve high-depth resolution which depends on the coherence
length of the source. This is the length over which a process or a wave maintains strict
phase relations; an ideal laser source, for instance, emits light with more than a few km
coherence length, while the coherence length of light emitted by a tungsten lamp could
be as short as 1 µm. Interference takes place only between events which happen within
the coherence length of the light source. Optical sources are now available with coherence
length below 1 µm. When this is combined with confocal microscopy, optical coherence
tomography improved depth resolution and sensitivity (Larin et al., 2002).
Figure 1.3: Cross section optical-coherence tomography (OCT) image from the skin on
the human finger tip. Image obtained in 0.5 s using a superluminescent diode of central
wavelength 793 nm and delivering 600 µW optical power to skin. Depth resolution is 15
µm (Podoleanu, 2005).
The Fig. 1.3, is a cross sectional image from the skin on the tip of a finger. Back-scattering
structures are resolved within a depth of 700 µm and which clearly displayed such as the
stratum corneum, sweat ducts and epidermis of the finger. Similar images, from different
types of tissue, are obtainable with modern OCT tools in fractions of a second and with
optical powers well below the maximum safety level. It is well known that coherence
length of the light source play a very important role to obtain high resolution of the
object. Using sources with extremely short coherence length, submicron depth resolution
is achievable even when the microscope objective is far away from the investigated target.
This is one of the most important feature of OCT, which explains the high level of
interest for OCT in ophthalmology. Confocal imaging was initially applied to obtain
high resolution images of the eye, OCT does not require contact in contrast to confocal
microscopy.

Chapter 1. Glucose Diagnostic Methods 1.4. Working Principle of OCT
Optical coherence tomography has also been extended for imaging higher scattering tis-
sues, such as skin, collagen, dentin and enamel. An increased interest is manifested in
the applications of OCT in endoscopy, with the development of specialized catheters to
accommodate different internal organs of the sample. OCT employs optical and infrared
waves and therefore it is dominated by diffraction which precludes algorithms for image
reconstruction used in X-ray or MRI. Sometimes analogies are made of OCT cross sec-
tion images with B-scan ultrasound images. However, ultrasound beams are longitudinal
waves, whereas the waves in OCT are transverse. It is true that similarity does exist
between the time taken for the ultrasound to propagate back and forth to the probe head
(giving distance for a known ultrasound velocity in tissue) and the time taken by the
optical waves in OCT to travel over a certain path length. However, whereas ultrasound
imaging is a time of flight technique, where time gating is used to display ordered time
events, in OCT the gating process operates in space, based on interferometry (Huang
and et al., 1991).
Optical coherence tomography fills the gap of the product of depth resolution and pen-
etration depth between confocal microscopy and ultrasound imaging. This product is
approximately 0.1 µm×500µm in confocal microscopy, 1 µm×3000µm in OCT and 50
µm×5000µm in high frequency ultrasound (Larin et al., 2002).
1.4 Working Principle of OCT
Different interferometer configurations can be used in optical coherence tomography. A

Michelson interferometer is shown in Figure 1.4, which is illuminated by an optical source
(OS). Light from the OS is divided into two beams by using a plate beam-splitter (BS).
Fig. (1.4) shows a different set-up, where the bulk BS is replaced by fiber beam-splitter
is known as directional coupler (DC). It has all the properties of a bulk beam-splitter
plus the advantage that the input and the output ports can easily be altered by moving
the fibre ends. Delivering light to the tissue and collecting the back-reflected light via an
optical fibre, which can be as thin as 0.125 mm. The two beams, reference and object,
are recombined by the beamsplitter BS in Fig. (1.4a) or by the directional coupler DC
in Fig. (1.4b) onto a photodetector. From interference theory, the photodetected signal
is defined as (Podoleanu, 2005)
√

P0 2π
iph =α O + R + 2 ORΠ cos( d) (1.2)
2 λ
where α is the photodetector responsivity, O and R are the target and reference mirrors
reflectivity, respectively. λ is the central wavelength of the optical source and P0 is the
power incident on the object. The optical path difference (OPD) between the two optical

Chapter 1. Glucose Diagnostic Methods 1.4. Working Principle of OCT
Figure 1.4: Optical-coherence tomography set-up: (a) represents Michelson interfer-

ometer and (b) represents in-fibre equivalent of the configuration in (a); OS, Optical
source; BS, Beam-splitter; OUT, Object under test; DC, single mode directional coupler
(Podoleanu, 2005).
paths in the two arms is d = 2 |lr − lo |. The interference amplitude depends also on the
degree of polarization which is described by the factor Π i.e. the degree of similarity of
the orientation of the electric fields in the two optical beams. In the above eq. (1.2), the
first two terms are time non varying which are used to determine noise, usually R O.
The third term represent the interference which is periodic and dependent on d and λ.
Reference mirror is moved each time by an extra λ/2 (which determine a round trip OPD
of λ), in order to do this the photodetected signal strength repeats itself. Hence, maxima
and minima are detected as the reference mirror is moved. The coherence length (lc ) of
the source is defined as
ln 2 λ2
lc = 4 (1.3)
π ∆λ
and thus the larger ∆l, the shorter lc and hence the better the OCT transverse resolution.
This formula is applicable for the Gaussian beam. Using the stepper motors, different
depths into tissue can be probed by changing the position of the reference mirror. If the
reference mirror is moved with a known speed then the third term (cosine) in above eq.
(1.3) will vary with time and allows the dc components of the signal to be filtered out and
the time-varying signal (ac component) amplified. If the λ/2 spatial interval is scanned
with a velocity v, then in time the peaks are separated by (λ/2)/v which is the inverse
of a frequency f. This means that by moving the reference mirror, the photodetected
signal oscillates with a frequency f=2ν/λ. This is called Doppler frequency because f is
identical in value with the Doppler shift experienced by the frequency of a wave reflected
from a moving mirror (Fujimoto, 2003).
A typical SLD has a bandwidth ∆λ=20 nm, i.e. lc ≈ 44µm which defines a round trip,
this leads to a depth pixel size of 22 µm in air, which in tissue, considering a refractive
index n of 1.4 gives 16 µm (Drexler, 2004; Fercher et al., 2003).

Chapter 1. Glucose Diagnostic Methods 1.5. OCT Imaging Modes
1.4.1 Why OCT?
• OCT uses wavelengths within the band of 600 nm-2000 nm where the main con-
stituents of the tissue, water, pigments, etc. exhibit low absorption.
• The main driving force behind OCT development is its high depth resolution. The
wider the optical spectrum line-width, the smaller the coherence length of the light
source and the better the depth resolution.
• In order to obtain an interference signal in OCT, a strict phase relationship is

required between the interfering waves. Multiple scattered events lose the phase
information. Therefore, only single scattered photons contribute to the interference
signal. Consequently, the maximum penetration depth in OCT is the depth from
which single scattered photons still originate. This depth is about 1.5 mm in skin
using wavelengths within the 800 nm band and about 2 mm when using 1300 nm
due to lower scattering at longer wavelengths (Pan et al., 1996).
• Photodetection at the interferometer output involves multiplication of the two op-

tical waves of the reference and sample arm, therefore the weak signal in the object
arm, backscattered or transmitted through the tissue, is amplified by the strong
signal in the reference arm. This explains the higher sensitivity of OCT when com-
pared with confocal microscopy, which for instance in skin can produce images only
to a depth of 0.5 mm (Rajadhaksha et al., 1999).
1.5 OCT Imaging Modes
3D information about an object can be obtained, the imaging system is equipped with
two scanning means: one to scan the object in depth and another to scan the object
transversely, usually composed of two orthogonal scanners. Depending on the order in
which these scans are operated and on the scanning direction associated with the line
displayed in the raster of the final image delivered, different scan planes are possible.
OCT systems using charge-coupled device (CCD) cameras or arrays of sensors or arrays
of emitters eliminate the need of scanning. The scanning terminology is illustrated in the
Fig. (1.5).
1.5.1 A-Scan based B-scan
B-scan images, analogous to ultrasound B-scan are generated by collecting many A-scan
for different and adjacent transverse positions as shown in Fig. (1.5). The lines in
the raster generated correspond to A-scan, i.e. the lines are oriented along the depth

Chapter 1. Glucose Diagnostic Methods 1.5. OCT Imaging Modes
Figure 1.5: Relative orientation of the axial scan (A-scan), en-face scan (T-scan), longi-
tudinal slice (B-scan) and en-face or transverse slice (C-scan) (Podoleanu, 2005).
coordinate. The transverse scanning means (operating along X or Y, or along the angle
θ with ρ constant in polar coordinates in Fig. (1.5), with X) which advances at a slower
pace to build a B-scan image. The majority of reports in the literature refer to this
method of operation (Drexler, 2004).
Figure 1.6: Different modes of operation of the three scanners in a flying spot optical
coherence tomography system (Podoleanu, 2005).
1.5.2 T-scan based B-scan
In this case, the transverse scanners (or scanner) determines the fast lines in the image;
each image line is a T-scan Fig. (1.5). This can be produced by controlling either the
transverse scanner along the X-coordinate, or along the Y-coordinate with the other
transverse scanner fixed, or controlling both transverse scanners along the polar angle θ
for a given ρ, with the axial scanner fixed. The example in the middle of Figure illustrates
the generation of a B-scan using several T-scans , where the X-scanner produces the T-

Chapter 1. Glucose Diagnostic Methods 1.6. Types of OCT Imaging
scans and the axial scanner advances slower in depth, along the Z-coordinate. This
procedure has a net advantage in comparison with the B-scan generated from several
A-scan as it allows production of C-scan, i.e. of OCT transverse (or en face) images for
a fixed reference path.
1.5.3 C-scan
C-scans are made from many T-scans along either of X, Y, ρ or θ co-ordinates, repeated
for different values of the other transverse coordinate, Y, X, θ or ρ, respectively, in
the transverse plane. The repetition of T-scans along the other transverse coordinate
performed at a slower rate is shown in Fig. 1.6, which determines the frame rate. In this
way, a complete raster is generated. Different transverse slices are collected for different
depths Z, either by advancing the optical path difference in the OCT in steps after each
complete transverse (XY) or (ρ, θ) scan, or continuously at a much slower speed than
the frame rate. Presently, no other technology can provide non-invasive and non-contact
in-vivo real time subsurface images with such a high depth resolution.
1.6 Types of OCT Imaging
Optical coherence tomography is a typical noninvasive imaging modality. OCT performs

the high-resolution, cross sectional tomographic images by measuring the backreflected or
backscattered light within internal microstructure of the biological tissues and materials.
The image resolution of 1-15 µm can be obtained. Non-invasive method must rely on
the intrinsic variation of tissue properties which differentiate tissue constitutes. However,
any physical property which alters the amplitude, phase, or polarization of the sample
beam that can be used to extract information about the diseases. Many mechanism are
used to find out the information from the sample. On the basis of it, new OCT imaging
modes have been demonstrated as
• Time Domain OCT
• Frequency Domain OCT
• Swept source OCT
• Longitudinal and En face OCT
• Functional OCT
• Polarisation sensitive OCT

• Doppler OCT
• Absorption OCT
• Elastic OCT
1.6.1 Time Domain OCT
The working principle of Time domain OCT is represented in fiber optic and free sapce
OCT Fig. 1.4, where an A-scan is produced by varying the optical path difference (OPD)
in the intererometer to output a reflectivity profile in the depth of the sample. En face
flying spot OCT belongs to the same category where a T-scan is produced by transversally
scanning the beam over the target maintaining the reference mirror fixed to generate a
reflectivity profile versus angle or lateral position (Fercher et al., 2003).
1.6.2 Frequency or Fourier or Spectral Domain OCT
Fourier domain OCT can provide a signal to noise ratio that is more than 20dB better
than the conventional Time domain OCT and sufficient sensitivity was demonstrated
by displaying video-rate images from the retina. Initially, Fourier domain OCT lagged
behind the coherence Time domain OCT due to the low speed and limited dynamic
range of CCDs. Progress in the field of high-speed, high-dynamic range CCDs and
photodetector arrays now makes Fourier domain OCT attractive. Fourier domain OCT
has two disadvantages:
1. focusing point by point in depth (a procedure called dynamic focus, often utilized in
the Time domain OCT) is not possible and therefore the interface optics is devised
with a large depth of focus, to accommodate the entire range of the A-scan, usually
a high numerical aperture objective to enhance the transverse resolution;
2. the optical spectrum of the interferometer output consists of symmetric spectral

terms, i.e. the same image results for positive and negative optical path difference
(OPD). For the latter, an initial adjustment of the OPD=0 (constructive interfer-
ence) outside the range of interest is required. This is not possible all the time,
especially when imaging moving thick organs or tissues.
Different methods have been devised to attenuate the symmetric terms in order to obtain a
correct image such as phase-shifting interferometry, or complex signal processing (Fercher
et al., 2003; Podoleanu, 2005).

1.6.3 Swept Source OCT
Recent progress in the fast tunability of laser sources has received interest in Swept source
OCT. In Swept source OCT, where a laser source is used in contrast to Time domain
OCT and Fourier domain OCT which both employ a wideband source. In order to obtain
similar depth resolution as in the Time domain OCT or Fourier domain OCT, the laser
frequency needs to be swept within an equivalent band to that of the broadband source
used in the Time domain OCT or Fourier domain OCT.
In the swept source, the achievable signal to noise ratio is similar to that of Fourier domain
OCT. Swept source OCT suffers from the same disadvantages as Fourier domain OCT,
as the modulation frequency of the interferogram is proportional to the absolute value of
the OPDs. Therefore, the origin of OPD=0 must be placed outside the depth range of
interest. The time required to tune the wavelength determines the time to produce an
A-scan. Tuning speeds of a few kHz have been achieved, which allows Swept source OCT
to compete with Time domain OCT and Fourier domain OCT in terms of speed (Fercher
et al., 2003).
1.6.4 Longitudinal and En face OCT
Longitudinal OCT: This is the main OCT technology used to image tissues and refers
to cross-section images (B-scan sections) in the plane: (depth axis) × (lateral or angular
axis), where the slice is constructed from many A-scans repeated for subsequent transverse
pixels (Fercher et al., 2003).
En face OCT: When using the set-up for en face OCT, lateral scanning is the fastest and
determines the line rate while the depth scanning to produce a B-scan image determines
the frame rate as shown in Fig. 1.6. In the C-scan regimen, one of the transverse
scanners determines the line rate and the other is slower, operating at the frame rate.The
depth scanning is the slowest in this case. It is more difficult to generate en face OCT
images than longitudinal OCT images as the reference mirror is fixed and no carrier is
generated. In T-scan based OCT images, a phase modulator is needed (Dobre et al.,
2005; Hitzenberger, 2003). A 3D image of normal human skin from a volunteer’s finger
tip is shown in Fig. 1.7. An image obtained form an healthy eye is shown in 1.8.
1.6.5 Functional OCT
OCT provides depth resolved information of reflectivity, phase and polarization of the
backscattered signal. This signal is intimately related to functional disturbances, which
usually precede morphological changes.

Figure 1.7: 3D display of in-vivo optical-coherence tomography image of normal human

skin from a volunteer’s finger tip. Volume size: 5mm × 4mm × 1mm (depth measured in
air). The arrow ED shows the direction of exploration of the 3D reconstructed volume
made from 40 en face slices acquired at 25 µm depth interval (Podoleanu, 2005).
Different versions of OCT systems provide functional informations, such as
• Polarization sensitive OCT
• Spectroscopic OCT
• Differential absorption OCT
• Doppler OCT.
1.6.6 Polarization Sensitive OCT
Polarization sensitive optical-coherence tomography takes into account the vectorial na-
ture of light waves (state of polarization). It can detect and quantify the polarization
properties of the tissue by changing in the polarization state of the backscattered light
beam. The information provided by polarization sensitive OCT images can be used to
identify birefringent structural constituents in the target tissue that are otherwise in-
visible to conventional OCT or other imaging techniques. A change in the polarization
sensitive OCT images of a tissue can be related to a change in the structure, function-
ality or integrity of the target. For instance, thermal injury denatures collagen in skin
and polarization sensitive OCT can sense changes in the collagen birefringent. Retinal
nerve fiber layer, cornea and dentin are birefringent in nature. The retinal nerve fiber
thickness is an essential parameter in the diagnosis of glaucoma and instruments based on
retardation measurements infer the thickness by assuming that the double-pass phase re-
tardation per unit of depth is the same throughout the eye fundus. Polarization sensitive
OCT studies have shown that the retinal nerve fiber layer double-pass phase retardation

Figure 1.8: Simultaneous indocyanine green (ICG) at left and en-face optical-coherence
tomography at right image from an healthy eye. Lateral size: 26o . Images collected at
40 s after ICG was released into the body. RPE, retinal pigment epithelium (Podoleanu,
2005).
per unit of depth (measured in degrees of rotation of the linear polarization vector/mm)
is not the same but varies with values between 0.18 and 0.37 degree/µm (Fercher et al.,
2003; Jiao et al., 2000).
Muscle, tendons and other body tissues contain collagen and elastin fibers that exhibit
birefringence when aligned in layers. Early in the development of OCT, polarization optics
were added to the basic OCT system to permit depth profiling of tissue birefringence. The
measurement of one-dimensional birefringence profiles was later extended to birefringence
imaging of normal and thermally damaged soft tissue. Further investigations using PS-
OCT revealed that the light backscattered from structures deep inside living skin is
highly depolarized. The depolarization was attributed to multiple scattering and to
single-scattering from non-spherical particles.
The dependence of the Stokes parameters of a random media on the density, sizes and
arrangement of scatterers suggests that the full characterization of the polarization state
of backscattered light may reveal information about tissue structure that is not appar-
ent in OCT images obtained using randomly polarized light. Although the ability of
polarization-sensitive OCT to enhance the contrast between healthy and pathological tis-
sue has not yet been established in clinical trials, local changes in the degree and state of
polarization of the sample beam have been observed in laboratory studies of thermally
damaged skin (Boer and Milneree, 2002; Born and E.Wolf, 1999; Cense et al., 2004; Hee
et al., 1992).

1.6.7 Doppler OCT
For nearly two decades, laser Doppler velocimetry has been applied in a variety of med-
ical studies, which include the measurement of blood flow in the skin, eye and other
organs. Because the laser has a long coherence length, interference between the static
and Doppler-shifted components of the light scattered in tissue occurs over an extended
optical path. Therefore, only coarse adjustment of the size and location of the sample
volume probed by the velocimeter is possible via selection of the emission wavelength of
the laser and the geometry of the probes. The idea of employing coherence gating to
define the sampling volume of a optical velocimeter was first applied to the measurement
of flowing particles for the study of fluid mechanics (Yazdanfar et al., 1997).
In Doppler OCT, the backscattering centre moves with constant velocity, then the OPD
varies linearly in time d = vt which leads to a frequency f = 2ν/λ, where ν is the
projection of the velocity vector along the interrogating beam direction. This frequency is
the Doppler shift suffered by the frequency of the object beam and appears as a frequency
bit when the object beam is mixed with the reference beam on the photodetector in the
OCT interferometer. Hence, Doppler OCT can be used to measure or monitor Brownian
motion and flows of biological liquids. In addition to laser anemometer, Doppler OCT
provides a depth resolved profile of the flow velocity in the vessel, with the resolution
determined by the coherence length of the source. Due to the fact that the scanning itself
shifts the frequency of the OCT signal, a challenging avenue in research is to produce the
OCT image and velocity map simultaneously (Fercher et al., 2003; Rollins et al., 2000;
Zhao et al., 2000).
1.6.8 Absorption OCT
Absorption OCT or differential OCT can be implemented using two channels OCT, each
channel operating on a different wavelength. One wavelength is chosen close to the
absorption peak of the constituent to be measured with the other wavelength exhibiting
low absorption. Such a technique was used to obtain depth resolved concentration of water
in the cornea by operating simultaneously at 1331 nm (where the water has relatively
low absorption) and at 1448 nm (where the water exhibits an absorption peak) (Fercher
et al., 2003).
1.6.9 Elasticity OCT
The sensitivity of optical coherence tomography to displacement of living tissue during

imaging is a troublesome problem which can lead to undesired blurring of images. OCT

Chapter 1. Glucose Diagnostic Methods 1.7. Application of OCT
elastography takes advantages of this sensitivity to quantify microscopic deformations

inside tissue induced by externally applied stress. The main aim of OCT elastography
or elasticity imaging is to measure the local variations of the stiffness inside a tissue
noninvasively. The primary quantitative measure of the stiffness is the shear elastic
modulus, which varies widely for different types of tissue. A number of disease processes,
including edema, fibrosis and classification, alter the elastic modulus of the extracellular
tissue matrix.
Possible medical applications of OCT elastography is that it can differentiate of hard and
soft tissues during biopsy. Using this technique imaging of arterial plaque tissues and
evaluation of wound healing is also possible. OCT elastography is also capable to solve
fundamental problems in biomechanics, such as the role of microscope deformations in
the development of the embryo (Fercher et al., 2003).
1.6.10 Tissue Clearing and Contrast Media for OCT
The propagation of light beam in the biological tissue depends upon the mismatch of the
refractive index between the extracellular fluid (ECF) and scatterers. OCT operates with
ballistic photons, i.e. photons which have been scattered only once. Therefore, in highly
scattering tissue, OCT exhibits a short penetration depth. The depth of light penetration
into highly scattering tissue can be improved by the application of bio-compatible and
osmotically active chemical agents. Similarly, to address the main source of scattering in
blood, which is the difference in refractive index between the cytoplasm of erythrocytes
and serum, dextran and an intravenous contrast agent have been investigated in-vitro
(Wiki-OCT, 2013).
1.7 Application of OCT
Optical coherence tomography was firstly proposed by Huang and et al. (1991) to image
the biological tissues and can provide the cross-sectional image within the micro-structure
layers of the tissues. OCT was performed in the vitro in the human retina and the
athrosclerotic plaque are known as a weekly scattering medium.
Using OCT the typical image resolution of 10-15 µm and upto 1 µm is possible. In the
OCT the image information is obtained in the electronic form and use for the processing
and analysis and storage. Finally OCT can be ultimately engineered to be compact
and low cost. Hence, OCT is suitable for the research and also useful in the industry
and clinical also (Fercher et al., 2003). Optical Coherence Tomography is a powerful
imaging technology in medical fields because it provides in situ visualization of tissue

micro-structure in real time without to need and excise and process a specimen as in
conventional biopsy and histopathology. OCT is already used as a medical imaging
method today and has great potential for future applications, as well. Since it works
with light, it can image tissue in-vivo without causing any long-term after effects (Fercher
et al., 2003; Wiki-OCT, 2013).
OCT can be applied in many diseases of the human body. Here, we introduces the
branchs of the medical filed where OCT give it tremendous performance as followings
(Fercher et al., 2003):
1. Ophthalmology
2. Dentistry
3. Dermatology
4. Gynaecology
5. Gerontology
6. Cardiology
7. Urology
1.7.1 Ophthalmology
OCT is most well-suited for imaging transparent tissue. The most obvious transparent
tissue on human is the eye and this is indeed the area where OCT is currently most
widely in use. The first in-vivo images of the human retina were created as early as
1993 and has progressed rapidly since then. Today, it is used to detect mascular holes,
edema and degeneration, thickness of the retinal nerve fiber layer and other symptoms of
eye disease, before actual damage to vision occurs (Wiki-OCT, 2013). In ophthalmology,
OCT has allowed to image of the back of the eye with 100 times better depth resolu-
tion than that using confocal scanning laser ophthalmoscopy. Anterior segment OCT
(AS-OCT) provides structural information of the cornea and anterior chamber without
contacting the eye, offering an ease of image acquisition and a considerable advantage
over ultrasound biomicroscopy (UBM). While it cannot be used to image deep structures
such as the ciliary body, as UBM can, as OCT has higher axial resolution. It is possible
to acquire high-resolution images of the sclera, angle and iris with as OCT imaging at
longer wavelengths 1.3 µm. High-resolution images of the anterior chamber angle can also
be obtained with 850 nm systems and this has led to the visualization of the trabecular
meshwork and Schlemm’s canal (Fercher et al., 2003).

The ability to measure glucose concentration through noninvasive approaches would im-
pact the treatment of diabetes significantly. Polarization-based optical approaches have
received considerable interest because of their potential medical applications. Glucose
is a chiral molecule which has the ability to rotate the plane of linearly polarized light,
commonly referred to as optical activity, as well as changing the refractive index of the
media, which therefore affects the overall scattering coefficient in a given media. The
magnitude of each effect is related to the concentration of glucose. Although most pre-
vious studies have reported on the use of polarimetry in the aqueous humor of the eye
because of its nonscattering nature, one would also expect that glucose concentration
could be measured in more turbid media such as tissue through a similar approach. This
study investigated how each of these effects is correlated to glucose concentration in a
physiological range for highly scattering biological media. Controlling of the optical prop-
erties of tissues are extremely important for many biomedical applications including the
development of noninvasive or minimally invasive glucose sensors as well as for therapy
and diagnostics of various diseases, such as cancer, diabetic retinopathy and glaucoma
(Drexler, 2004; Fercher et al., 2003; Wiki-Blood, 2013).
1.7.2 Dentistry
OCT provides effective imaging method in investigation of optical and structural prop-
erties of dental tissue for example in diagnosis of caries, measurement of tooth colour
etc. It gives information about the oral cavity within 2 to 3 mm of tissue. Polarization
sensitive OCT (PS-OCT) is able to find images of dental and human teeth. A field where
polarization dependent backscattering might play an increasing important role in dental
OCT. Human teeth consist primarily of enamel, dentin and pulp. The pulp of the tooth
is made of semitransparent dentin with micrometer-sized dentinal tubules, radiating from
the pulp cavity towards the periphery the outer surface of the tooth is covered by a thin
and transparent layer of enamel consisting of micro crystal oriented normally to the sur-
face. PS-OCT can provide additional information related to the mineralization status
and the scattering properties of the dental materials (Fercher et al., 2003; Wang et al.,
2002; Wiki-OCT, 2013).
1.7.3 Dermatology
Optical coherence tomography is a non-invasive imaging technique, which has previously

demonstrated potential for use in dermatology. Using OCT many part of the human skin
can be imaged and can get information about the diseases. The layered structure and
appendages of skin were apparent in conventional OCT images and correlated well with

corresponding histology (Wiki-OCT, 2013).
1.7.4 Cardiology
OCT sustains high resolution capability, it is useful in diagnosis of plaques in coronary

vasculature. It is able to differentiate between stable plaques and unstable plaques. Heart
attack is caused by rupture of the cholesterol filled in lesions plaques. When a plaque
bursts, a blood clot forms and blocks the heart blood vessel. To image the plaque OCT
is a powerful technology (Wang et al., 2002).
1.7.5 Urology
The different layer of urinary bladder and ureter can be resolved with OCT. Stone problem
can also be solved by measuring the exact size of stone in kidney and this can be done
by using high resolution and 3D OCT (Wiki-OCT, 2013).
1.7.6 Optical Catheter or Endoscope
In the field of endoscopy, OCT prevails as the only technology capable of high resolution
imaging with better than 15 times depth resolution than high frequency ultrasound.
New imaging technology brings not only new information to the clinician, but with it
the requirement of interpretation. OCT is no exception in this respect; curvature of the
tissue, coupled with the variation of different optical properties from layer to layer, organ
movement and the specific way of ray scanning make the correct representation of the
OCT image challenging.
Another common application, due to the possibility of using OCT via an optical catheter
or endoscope, is using OCT to acquire images from inside the body. Imaging is performed
via rotating, pushing/pulling the tube, or other optical or mechanical mechanisms, gener-
ating transverse scans from inside vessels or hollow organs. There is considerable interest
in using OCT for the detection of cancer symptoms in the esophagus, stomach and colon,
as unlike biopsy it would enable cancer detection without requiring the excision of tissue.
Since the performance of OCT is often not quite at the level required for a stand alone
diagnosis, there is also ongoing research into using OCT to assist biopsy, by picking a
good location for excision (Fercher et al., 2003; Wiki-OCT, 2013).

Chapter 1. Glucose Diagnostic Methods 1.8. Optical Properties
1.8 Optical Properties
The optical properties play an important role in the investigation of diseases. Various
diseases can be investigated easily if the optical properties are known. The optical prop-
erties of the human skin correlates with skin anatomy. The Human skin are basically
classified in the 3 components or layers: 1. the most superficial layer is called the epi-
dermis (Epidermis-above), 2. the middle layer (Dermis) and 3. the deepest layer (the
subcutaneous tissue) which is placed just above the muscles and other deeper structures
(Cheong et al., 1990; Maruo et al., 2003; Wiki-Skin, 2013).
1.8.1 Epidermis
The epidermis is the most superficial layer of the skin. When discussing either thick or
thin skin (or glabrous and hairy skin), it is mainly the thickness of the epidermis which
is meant, but also the innervation and the presence of hair varies between the skin types.
The thickness of the other layers will not be solely dependent on whether the skin is
thick or thin, often the thick is also called glabrous skin where no hairs are present, such
as palms and soles. The thickness of the entire epidermis varies through out the body.
In hairy skin the epidermis is typically 80 to 100 µm thick and in the thicker glabrous
skin the epidermis can be as thick as 500 µm. However, these thickness can vary a lot
depending on the placement on the body, gender, age and environmental effects (Larin
et al., 2003; Wiki-Skin, 2013).
1.8.2 Dermis
Unlike the epidermis the dermis is vasculized and highly innervated by nerves. Besides
the blood vessels, nerve fibers and connective tissue the dermis also contains sweat glands
and ducts, the sweat ducts leads from the dermis through the epidermis and onto the
skin surface. The entire dermis is typically 1-3 mm thick (Wiki-Skin, 2013).
1.8.3 Subcutaneous
The subcutaneous tissue separates the skin from the deeper structures and organs such as
muscles like the dermis, the subcutane tissue are vasculized and contains high amounts of
connective tissue, the connective tissue of the subcutane and the dermis are interwoven
making it difficult to clearly seperate the two layers from each other (Larin et al., 2003;
Wiki-Skin, 2013).
The human skin has several functions such as protection, sensation, creation of vitamin

Chapter 1. Glucose Diagnostic Methods 1.8. Optical Properties
D3 and storage of different substaneous. The epidermis serves as an effective first barrier
against the environment. It both keeps necessary substances within the tissue, such
water and ions and the sebum secreted helps the skin being water resistant and inhibits
bacteria growth. The dermis is perfused by a number of blood vessels, these vessels
originate from plexuses in the subcutaneous tissue. The subcutaneous tissue has several
functions especially the adipose tissue. Due to the non-planar surface of the skin any
incident collimated beam of light will not produce either a collimated reflection nor a
collimated beam entering the skin. Instead both diffuse reflection and refraction will
occur. In the spectrum 250-3000 nm, 4-7 % of a normal incident radiation will always be
reflected at the skin surface.
The refractive indies of the superficial skin layers was found in, therefore a refractive
index of epidermis was set to 1.34 and for dermis it was set to 1.41. For the subcutaneous
tissue the refractive indexes is 1.4-1.45 for the blood the refractive index is 1.4 (Wiki-Skin,
2013).
1.8.4 Human Blood
Human blood consists of solid (scatterers) and liquid (Plasma or serum). Using OCT, we
are able to measure the concentration of glucose in human subjects when the refractive
index mismatch occurs between ECF and scatteres of the medium (Larin et al., 2002;
Wiki-Blood, 2013). The concentrations of the different cells is shown in Fig. 1.9
Figure 1.9: Concentrations of different cells in human blood is shown (Poddar et al.,
2008).

Chapter 1. Glucose Diagnostic Methods 1.9. Skin Structure
1.8.5 Intralipid
The water solution of intralipid is frequently used for the fabrication of biotissue phan-
toms. Intralipid is a poly-dispersed suspension of almost spherical particles of about 3.5
µm mean diameter suspended in glycerine and water solution. The particles are soybean
oil droplets covered with a 2.5-5.0 nm thick lipid membrane. In medicine this substance
is used for intravenous feeding. The optical properties of intralipid are well known. How-
ever some fluctuations are possible among different manufacturers (Cheong et al., 1990;
Flock et al., 1992; Staveren et al., 1991).
1.9 Skin Structure
The complex structure of skin is shown in the Fig. 1.10 which illustrates the different skin
layers like the epidermis, dermis and subcutaneous tissue (Larin et al., 2003; Wiki-Skin,
2013). However, for many of the problems in biomedical optics the skin can be considered
as a single-, two- or three-layer medium using the effective averaged parameters for each
layer. The propgation of the light beam in tissue sample is shown in Fig. 1.11 which
gives information about different scattering within sample.
1.10 Scattering Medium
A scattering medium is that the intensity distribution of the ‘shadow’ (of objects in the
beam) is disturbed as the photons are deviated from their straight trajectory. However,
some of the photons will not be scattered and will continue to propagate along straight
lines these are called ‘ballistic photons’. As we delve deeper into the medium, the num-
ber of unscattered photons drops exponentially until, at significant depths, the multiply
scattered photons will overwhelm the ballistic signal. In most real mediums, ballistic
photons are further consumed by absorption. For ballistic light imaging using 800 nm
radiation within the limit of permissible intensities on living patients, (Hee et al., 1992)
have estimated that the ballistic signal will drop below the shot noise threshold after 36
mean free paths (MFP) in the tissue. This corresponds to a tissue depth of 4 mm, as
the MFP is about 100 µm. Turbid media and biological tissues are highly forward scat-
tering, denoted by the anisotropy parameter g which is the mean cosine of the scattering
angle. Since g = 0.7 − 0.9, most photons will only be slightly deviated from their original
direction after a scattering events (Bass et al., 1995).
One might find a significant number of photons only slightly deviated and thus follow a
‘snake like’ path about the ballistic direction where the transport scattering coefficient

Chapter 1. Glucose Diagnostic Methods 1.10. Scattering Medium
Figure 1.10: Different layers of the human skin is shown (Wiki-Skin, 2013).

Chapter 1. Glucose Diagnostic Methods 1.10. Scattering Medium
Figure 1.11: Propagation of the light beam in the biological tissue is shown (Bashkatov
et al., 2003).
Figure 1.12: The change in the intensity of incident signal after scattering medium is
shown (Poddar et al., 2008).
0
µs = µs ∗ (1 − g). This leads one to expect that snake-like light can be detected corre-
sponding to 4 cm of tissue. After propagating more than two or three transport lengths,
most photons are heavily scattered and may be described as diffuse. Figure 1.12 pro-
vides a sample of different photon trajectories through a scattering medium, rendering a
modification of the temporal impulse. A detailed theoretical analysis using Monte Carlo
simulation of multiple scatterer in tissue is provided with experimental analysis. Several
models of tissue scatterers have been developed involving fractals and discrete particles.
Additionally, studies have been performed to determine the dependencies of OCT pa-
rameters on the optical properties of tissue, especially to account for the signal-degrading
multiple scatterer. Due to the burden, some nature of analytical methods for more than
a few mm of tissue, transport theory results have been invoked.

Chapter 1. Glucose Diagnostic Methods 1.11. Measuring Site
1.11 Measuring Site
To obtain better quality spectra of skin tissue, one effective tactic is to improve the S/N
of an instrument. However, the S/N of an instrument is one of the important factors of
a total measurement system. Therefore, other factors must be considered as well. The
selection of the measuring site of human tissue that provides sufficient information about
blood glucose is also important. When a NIR spectrum of skin tissue is measured, the
existence of the stratum corneum is a major problem. To avoid the interference from
the stratum corneum, NIR spectra of oral mucosa using reflection optics based on an
ellipsoidal mirror because the oral mucosa does not contain stratum corneum. These
studies suggested that avoiding the influence of the stratum corneum was effective to
obtain a good performance for a blood glucose assay (Schmitt, 1999).
The dermis tissue of a forearm is chosen for blood glucose assay using the first overtone
region. A forearm skin tissue consists of three layers, i.e., epidermis, dermis and subcu-
taneous tissue. The epidermis contains the stratum corneum and capillary vessels are
not developed sufficiently in it. This means that the epidermis, especially the stratum
corneum, does not contain useful information about blood glucose content and acts as
interference when the skin tissue spectra are measured for a blood glucose assay. The
subcutaneous tissue is composed mainly of fatty tissues. This tissue also does not provide
information about blood glucose. On the other hand, there are well-developed capillary
vessels in the dermis and blood glucose is easily transferred to the dermis tissue due to
its high permeability. Thus, the glucose content in the dermis is assumed to correlate
with the blood glucose content in the same way as that in the interstitial fluid. If the
dermis spectra could be measured selectively, the interference noise originating from the
epidermis or subcutaneous tissue would be removed (Larin et al., 2003; Schmitt, 1999).
1.12 Role of Glucose
The changes in the OCT signal with increasing blood glucose concentration is highly
sensitive compared to the OCT sensitivity to changes in N a+ concentration due to NaCl
injections. In normal conditions, diurnal variations in N a+ concentrations usually does
not exceed 1 mM (18 mg/dl), while clinically important variations in blood glucose con-
centration would exceed 2 mM (36 mg/dl). Therefore, change in OCT signal is noticeable
due to variation of glucose only and not noticeable by the fluctuations of other biochemical
and physiological variables (Larin et al., 2003; Varley, 1969).

Chapter 1. Glucose Diagnostic Methods 1.13. Electrolytes
1.13 Electrolytes
Sodium and potassium are the principle cations of clinical interest. Chloride and bicar-
bonate being the anions. Other electrolytes in serum include calcium, magnesium cations,
phosphate, sulphate, organic acids and protein as anions. Sodium and potassium earlier
determined by precipitation of their salts and subsequent calorimetry. The procedures
were very time consuming, laborious and open to many errors. Flame photometry (Flame
omission spectroscopy) is the method most commonly used for their determination (Larin
et al., 2004, 2003, 2002; Varley, 1969).
1.13.1 Flame Photometry
Alkali metals, when raised to a sufficiently high temperature will absorb energy form
the source of heat and be raised to an excited state in their atomic form. As each
atom cools to its original unexcited state it will reemit the absorbed energy by way of
radiation at specific wavelength, some of which is in the visible region. Therefore, if an
alkali metal in solution is aspirated into a low temperature flame in an aerosol form,
it will alter excitation by the flame, emit a discrete frequency which is isolated by an
optical filter. The emission is proportional (for low concentrations only) to the number
of atoms returning to the ground state, which is, in turn, proportional to the number
of atoms excited i.e. the concentration of the sample. Photodetector placed behind the
filter converts it to an electric current of the proportional amount which is measured
(Wiki-PFP, 2013).
Type of Flame Photometers:
• Direct Reading Instruments: Here the intensity of the emitted light is compared to
that obtained from standards treated similar to the sample.
• Internal Standard Instruments: The light intensity emitted form the element under
investigation is compared with that from an element which acts as an internal
standard.
Other methods which are used for serum electrolyte determinations include atomic ab-
sorption spectrophotometry and ion specific electrodes. As with many methods the in-
troduction of automation has improved the speed and accuracy of each of the methods
(Wiki-PFP, 2013).

Chapter 1. Glucose Diagnostic Methods 1.14. Methods for Non-invasive Glucose Monitoring
1.13.2 Atomic Absorption Spectroscopy
It is a modification of the flame photometer in that it measures the amount of light

absorbed by the nebulized sample while moving from ground state to excited state (Wiki-
PFP, 2013).
1.13.3 Ion Selective Electrodes
The potentiometric techniques measure the potential difference between two electrodes.
Specific electrodes are used for the determination of sodium, potassium and chlorides. The
requirement is for an ion selective membrane to separate the solution of known activity
from the detecting system. The membrane consists of special glass, a disk of crystalline
material or an organic ion exchanger saturating a water-immiscible solvent held in a gel
or plastic. The sodium electrode is sensitive to changes in sodium ion concentration and
potassium electrode is sensitive to changes in potassium ion concentration. Normal value
of electrolytes concentration in human body is given as sodium (135 - 145 mMol/L),
potassium (3.5-4.5 mMol/L) and chloride (95-105 mMol/L) (Wiki-PFP, 2013).
1.14 Methods for Non-invasive Glucose Monitoring
Different approaches have been used for the monitoring of the blood glucose using OCT
as followings:
1.14.1 Differential phase-contrast optical coherence tomogra-

phy (DP-OCT)
Differential phase-contrast optical coherence tomography (DP-OCT) was recently in-

troduced in a bulk optics system by Larin et al. (2004) and in a fiber interferometer.
Although, conventional OCT is based on the detection and analysis of the intensity
of backscattered optical radiation, DP-OCT utilizes the phase information obtained by
probing a sample simultaneously with two common-path low-coherence beams. Varia-
tions in the sample refractive index are reflected in the phase difference ∆φ between these
two beams. The DP-OCT technique is capable of measuring Ȧ-scale path-length changes
between the beams associated with the phase difference as ∆φ = λ/4φ∆φ in clear and
scattering media. The highly sensitive and accurate noninvasive detection of analyte con-
centration in turbid media is particularly relevant to the development of a blood glucose
biosensors. Monitoring of changes in tissue scattering as a function of blood glucose con-
centration (Variation of glucose concentration in the extracellular space produces changes

in the refractive-index mismatch between the extracellular fluid and the scattering centers
and, therefore, affects the tissue scattering properties) (Larin et al., 2002).
1.14.2 Phase Sensitive-OLCR
Phase sensitive optical low coherence reflectometry (PS-OLCR) based method can be
used for the measurement of the blood glucose. Phase shift versus glucose concentration
obtained in turbid media (aqueous suspension of polystyrene microspheres) with scatter-
ing coefficients similar to those of human tissues in the NIR spectral range (Larin et al.,
2004).
Figure 1.13: Phase shift versus glucose concentration obtained in turbid media (aqueous
suspension of polystyrene microspheres) with scattering coefficients similar to those of
human tissue in the NIR spectral range: squares, µs ∼ = 50cm−1 ; circles, µs ∼
= 100cm−1
(Larin et al., 2004).
Fig. 1.13 represents a typical dependence of the phase shift on glucose concentration in an
aqueous suspension of polystyrene microspheres of fixed concentrations. The scattering
coefficients of the two phantoms were 50 and 100 cm−1 . The concentrations of polystyrene
microspheres were chosen according to the calculations performed on the basis of Mie
scattering theory and were equal to 0.8% and 1.6% (w/W). Five glucose concentrations
in the range 2000 mM with a 20-mM increment were measured in these experiments. The
results obtained were similar to those found in clear-media studies (solid line), suggesting
the applicability of this technique to the sensing and monitoring of turbid samples (Larin
et al., 2004).
Limitation of PS-OLCR: Temperature control of the sample under study is important

in PS-OLCR measurements. Figure 1.14 demonstrates the dependence of the refrac-

tive index of water on temperature over the range from 0 − 1000 C. Evidently, small
fluctuations in the sample temperature i.e. 10 C can reduce the accuracy of phase-shift
measurements obtained with PS-OLCR (Larin et al., 2002).
Figure 1.14: Index of refraction of water as a function of temperature measured at

λ=1.01µm (prominent spectral line of mercury) (Larin et al., 2004).
The PS-OLCR method applied to the detection of glucose concentration has the level
of accuracy that is required for clinical studies (1 mM). PS-OLCR has the potential for
use in the noninvasive, sensitive and accurate monitoring of analyte concentrations both
in clear and turbid media. The influence of several potential obstacles on the sensitivity
and specificity of the PS-OLCR method for in-vivo monitoring of analytes concentrations
in highly scattering tissues (e.g., birefringence of tissues, stability of specular reference
points and tissue temperature) will be possible (Larin et al., 2004).
1.14.3 OCT Signal slope and Scattering Coefficient
Larin et al. (2003) measured the concentration of glucose using slope of OCT signal
(squares with SD bars) and scattering coefficient (line) considering Mie theory of scatter-
ing versus glucose concentration in the aqueous suspension of polystyrene microspheres
is shown in Fig.1.15.
From this Fig.1.15, it is clear that slope of the OCT signal decreases with increasing
glucose concentrations. To validate the experimentally measured dependence of the re-
fractive index on glucose concentration, we used a conventional OCT system to study

Figure 1.15: Slope of OCT signal signal (squares with SD bars) and scattering coefficient
(line) calculated with the Mie theory of scattering versus glucose concentration in the
aqueous suspension of polystyrene microspheres (Larin et al., 2004).
the dependence of OCT signal slope as a function of glucose concentration in an aqueous

suspension of polystyrene microspheres. The principle of operation and the algorithm of
mathematical processing of signals that were obtained with this conventional OCT sys-
tem λ = 1.3µm (Larin et al., 2004, 2002). One can see from this Fig. 1.15 that the linear
decrease of the OCT signal slope is in good agreement with the theoretically calculated
decrease of the scattering coefficient with glucose concentration.
1.14.4 Deconvolution Method
OCT signals obtained for different concentrations of glucose are deconvoluted and shown
in the inset of Fig.1.16. The deconvoluted signals contain more information about the
light scattering. With increasing concentration the area as well as the tail part of the sig-
nal increases. This is a clear indication of increasing value of reduced scattering coefficient
with decreasing glucose concentration. The measured scattering coefficients obtained af-
ter Monte-Carlo simulation are depicted as a semilog plot is shown in Fig. 1.16(Poddar
et al., 2008).
The curve (solid circles) of Fig. 1.17 exhibits the nature of reduced scattering coefficient
for different concentrations of glucose demonstrates a near logarithmic nature. In hypo-
glycemic region it shows a sharp change while in the hyper-glycemic region it has smaller

Chapter 1. Glucose Diagnostic Methods 1.15. PS-OLCR Based Analytes Measurements
50mg/dl
Deconvoluted Signal (a.u.)

100mg/dl
150mg/dl
0.9 200mg/dl
0.6
1.2 0.3
0.0
OCT signal (a.u.)
0 50 100 150
0.8 scan distance ( m)
W ater
50mg/dl
0.4 100mg/dl
150mg/dl
200mg/dl
0.0
0 20 40 60 80 100
scan distance ( m)
Figure 1.16: OCT signal for different glucose concentrations. For higher concentrations
the amplitude of the signal decreases while the width of the curve increase. (Inset)
Deconvoluted signals. The OCT signal obtained for water is taken as the source coherence
function. The deconvoluted signals are normalized (Poddar et al., 2008).
slope leading to less accuracy. A similar nature is also observed while measuring the area
of the deconvoluted signal (open circles) is shown in Fig. 1.17. This also experiences
a near logarithmic behavior. Repeated measurement shows that the slope of the curves
is a constant. Also, addition of more or less intralipid does not change this behavior.
However, in order to find the value of unknown concentration of glucose, one needs an
initial value at particular concentration of glucose. Since, the slope remains a constant,
this method could be better solution for non-invasive, non-contact, in-vivo monitoring
of blood glucose concentration. In order to realize this technique as a clinical tool more
efforts are required (Poddar et al., 2008).
1.15 PS-OLCR Based Analytes Measurements
The application of novel polarization-maintaining (PM) fiber-based dual-channel phase-

sensitive optical low-coherence reflectometer (PS-OLCR) is used for the highly sensitive
detection of analyte concentrations in clear and turbid tissue phantom (Larin et al., 2002).

Chapter 1. Glucose Diagnostic Methods 1.16. Conclusions
35
HYPER
20
HYPO NORMAL LEVEL OF
GLYCEMIC BLOOD GLUCOSE GLYCEMIC
30
15
Area (a.u.)
25
'
s
10
20
5
15
20 40 60 80 100 120
Glucose Concentration (mg/dl)
Figure 1.17: Semilog plot of reduced scattering coefficient and curve area with glucose
concentration. Data were extracted from Monte Carlo simulation and fitted to the ex-
perimental data. The error bars were obtained after 25 measurments (Poddar et al.,
2008).
1.16 Conclusions
This chapter reviews various methods being tested by different research groups all around
the world. The advantages and limitations of the methods are also discussed. The review
gives the current situation, that the accepted methods are either invasive or minimally
invasive. Although, non-invasive methods provide good results, but needs various cal-
ibrations and testing before being marketed. On the other hand, Optical coherence
tomography looks like a promising tool if measurement on polarization sensitivity is also
included. In view of this, we have undertaken the task of glucose level monitoring by
using optical coherence tomography technique.


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Chapter 1. Glucose Diagnostic Methods 1.1.

1.2 Chemical Methods

1.2.1 Invasive Method

Glucose (C6 H12 O6 ) + O2 + H2 O −

Figure 1.1: Anomers of D-glucose in an aqueous solution (Poddar et al., 2008).

1.2.2 Minimally Invasive Method

1.2.3 Continuous Glucose Monitoring (CGM)

CGM provides additional temporal information, such as trends, magnitude, duration

1.2.4 Self-Monitoring of Blood Glucose (SMBG)

SMBG levels is highly recommended by the American Diabetes Association as an integral

patients use conventional glucometers, the number of daily measurements is limited by

1.3 Physical Methods (Noninvasive)

Noninvasive determination of glucose can be classified in two broad categories as meth-

1.3.1 Near Infrared Spectroscopy (NIR)

corresponding to water displacements or changes in its intrinsic absorption. Changes in

1.3.2 Mid-Infrared Spectroscopy (Mid-IR)

Mid-infrared (Mid-IR) spectroscopy is based on light in the 2,500-10,000 nm spectrum

1.3.3 Temperature-Modulated Localized Reflectance

1.3.4 Raman Spectroscopy

1.3.5 Polarization Changes

1.3.6 Ultrasound and Photoacoustic Techniques

1.3.7 Fluorescence Technology

1.3.8 Thermal Spectroscopy

1.3.9 Ocular Spectroscopy

1.3.10 Bio-impedance Spectroscopy

1.3.11 Electromagnetic Sensing

Similar to impedance spectroscopy, this technique detects the dielectric parameters of

1.3.12 Fluid Harvesting Technique

minimally invasive (Mitragotri et al., 2000).

1.3.15 Optical Coherence Tomography (OCT)

Optical coherence tomography (OCT) is based on Michelson interferometer which use

1.3.16 Construction of OCT

for high-resolution ranging and characterization of optoelectronic components. The first

1.4 Working Principle of OCT

Different interferometer configurations can be used in optical coherence tomography. A

Figure 1.4: Optical-coherence tomography set-up: (a) represents Michelson interfer-

1.4.1 Why OCT?

• In order to obtain an interference signal in OCT, a strict phase relationship is

• Photodetection at the interferometer output involves multiplication of the two op-

1.5 OCT Imaging Modes

1.5.1 A-Scan based B-scan

1.5.2 T-scan based B-scan

1.6 Types of OCT Imaging

Optical coherence tomography is a typical noninvasive imaging modality. OCT performs

• Time Domain OCT

• Frequency Domain OCT

• Swept source OCT

• Longitudinal and En face OCT

• Polarisation sensitive OCT

1.6.1 Time Domain OCT

1.6.2 Frequency or Fourier or Spectral Domain OCT

2. the optical spectrum of the interferometer output consists of symmetric spectral

1.6.3 Swept Source OCT

1.6.4 Longitudinal and En face OCT

1.6.5 Functional OCT

Figure 1.7: 3D display of in-vivo optical-coherence tomography image of normal human

Different versions of OCT systems provide functional informations, such as

• Polarization sensitive OCT

• Differential absorption OCT

1.6.6 Polarization Sensitive OCT

1.6.7 Doppler OCT

1.6.8 Absorption OCT

1.6.9 Elasticity OCT