Optical Coherence Tomography

Chapter 1
INTRODUCTION
Optical coherence tomography (OCT) is an established medical imaging technique that
uses light to capture micrometer-resolution, three-dimensional images from
within optical scattering media (e.g., biological tissue). Optical coherence tomography
is based on low-coherence interferometry, typically employing near-infrared light. The
use of relatively long wavelength light allows it to penetrate into the scattering
medium. Confocal microscopy, another optical technique, typically penetrates less
deeply into the sample but with higher resolution.
Depending on the properties of the light source (superluminescent
diodes, ultrashort pulsed lasers, and supercontinuum lasers have been employed),
optical coherence tomography has achieved sub- micrometer resolution (with very
wide-spectrum sources emitting over a ~100 nm wavelength range).
Optical coherence tomography is one of a class of optical tomographic techniques.
A relatively recent implementation of optical coherence tomography, frequencydomain optical coherence tomography, provides advantages in signal-to-noise ratio,
permitting faster signal acquisition. Commercially available optical coherence
tomography systems are employed in diverse applications, including art conservation
and diagnostic medicine, notably in ophthalmology and optometry where it can be used
to obtain detailed images from within the retina. Recently it has also begun to be used in
interventional cardiology to help diagnose coronary artery disease. It has also shown
promise in dermatology to improve the diagnostic process.

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Chapter 2
BASIC PHYSICAL PRINCIPLES
Clinical examination using the slit lamp has been used for several years as the main
instrument for retinal structural assessment. Meanwhile, many other imaging techniques
have been developed to examine cross-sectional retinal morphology. The confocal
scanning laser ophthalmoscope (cSLO) forms retinal images by sequentially collecting
reections from laterally and longitudinally well-dened retinal volumes. Several cSLO
images taken with sequential focal depths can generate three-dimensional information
on the distribution of retinal reectivity for topographic and tomographic assessments
(Huang, 1999). The longitudinal resolution of a cSLO, however, is limited to!300mm
due to the available numerical aperture through the pupil and ocular aberrations
(Bartsch and Freeman, 1994). Cross-sectional measurements of the retina can be
achieved in the clinical setting as well by the instrument coined retinal thickness
analyzer (RTA). The RTA employs the principle of optical triangulation to provide
direct measurement of the retinal thickness with an estimated accuracy of 2030mm
(Zeimer et al., 1989). The instrument projects a narrow slit of 543nm HeNe laser light
onto the retina and calculates the distance between the reections that correspond to the
vitreoretinal and chorioretinal interfaces. Although recent advances in this
instrumentation have enabled rapid multiple optical sectioning of neighboring retinal
regions to generate a retinal thickness map (Zeimer et al., 1996), information is
restricted to fundus (macular) regions of 2"2mm and limited qualitative data can be
extracted from such imaging methodology. Optical coherence tomography (OCT) is
based on the imaging of reected light. But unlike a simple camera image that only has
transverse dimensions (left/right, up/down), it resolves depth. The depth resolution of
OCT is extremely ne, typically on the order of 0.01mm or 0.4 thousandth of an inch.
This provides cross-sectional views (tomography) of internal tissue structures similar to
tissue sections under a microscope, without disturbing the tissue as in histology. Thus,
OCT has been described as a method for non-invasive tissue biopsy.

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Figure 2.1 : The optical coherence tomography beam is scanned across retina
In OCT, a beam of light (typically 8001400nmwavelength in the near infrared) is
scanned across the tissue sample. The OCT system then collects the reflected light and
measures its time-of-flight delay. Light reflected from deeper layers have longer
propagation delays than that reflected from more superficial layers . The amplitude of
reflected light can be plotted against delay (Fig. 2.2) to demonstrate tissue reflectivity at
successively deeper levels of tissue penetration along the axis of beam propagation.
This is called an axial scan (A-scan). As the OCT probe beam is scanned across a
sample, many A-scans are acquired to form an image (Fig.2.3). A color or gray-scale is
used to represent the signal amplitude.

Figure 2.2 : An axial scan (A-scan) of the retina. The amplitude of reflection on
a decibel (dB) scale is plotted against depth.

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Figure 2.3 : An optical coherence tomography cross-sectional image (grayscale

image) is built up from many A-scans (red plot lines)

Chapter 3
BLOCK DIAGRAM
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Figure 3.1 : Block Diagram

Chapter 4
WORKING
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Ultrasound imaging and RADAR are also reflectometry- based imaging methods.
Because OCT employs light, several advantages are gained. The wavelength of light (!
0.001 mm) is shorter than that of ultrasound (!0.1 mm) and radio wave (410 mm).
Therefore the spatial resolution of OCT is much higher. And unlike ultrasound imaging,
OCT does not require probe-tissue contact or an immersion fluid since light passes
through the airtissue interface easily. Because light travels very rapidly (3"108 m/s), it
is not possible to directly measure the time-of-flight delay on a small spatial scale. The
micron-scale resolution of OCT is achieved by comparing the delays of sample
reflections with the known delay of a reference reflection in an interferometer. The
classic OCT system employs a low-coherence fiber-optic Michelson interferometer
(Huang et al., 1991). Interferometry measures the effect of combining 2 light waves.
Low coherence means that the system employs a wide range of wavelengths. We will
explain the concepts of interferometry and coherence separately.
The interferometer has source, sample, reference, and detector arms all centered
on a 50/50 fiber coupler. Output of the superluminescent diode (SLD) light source is
launched into the source arm fiber and split by the coupler into the sample and reference
arms. Sample and reference reflections are recombined at the coupler and produce
interference. This interferometric signal is converted from light to electrical current by a
photodetector, processed electronically, and transferred to computer memory. To
understand how the Michelson interferometer works, let us start with the simple case
where the sample arm reflection comes from a simple mirror surface and the light
source emits only one wavelength. Think of the sample and reference reflections as 2
waves of light. The coupler partially transfers theses waves to the detector arm, where
they combine and produce an interference signal. Interference can be thought of as the
addition of the amplitude of two waves. When the two waves are in phase (lined up
peak to peak), they interfere constructively, forming a peak in the interference
waveform. When the 2 waves are exactly out of phase (lined up peak to trough), they
interfere destructively, forming a trough in the interference waveform. As the reference
mirror is moved, the phase of the reference wave changes, producing a sinusoidal
interference signal. As the reference mirror moves through 12 cycle of the source

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wavelength, the roundtrip delay of the reference wave varies by one wavelength and the
interference signal goes through one cycle of sinusoidal oscillation.
To resolve the delay of sample reflections, the OCT system uses a light source that
has a wide range of wavelengths (low coherence). When the interference signals are
added together over the range of wavelengths, the interferometric oscillation fades as
the delay mismatch between the reference and sample reflections increases . To
understand why this occurs, look carefully at the phase relationship between the
wavelength components . When the delay mismatch is near zero (center of waveforms),
the interference signals from all wavelength components have the same phase (peaks
and troughs lined up) or are coherent. This adds up to large interferometric
modulation (see large peaks and troughs in the center of the rightside waveform). When
the mismatch is large (away from the center of the waveforms), the interference
waveforms vary widely in phase over the wavelength range (peaks and troughs not lined
up) and add up to near a flat line. The summed interference signal forms a wave pulse.
In an OCT system, the pulse is demodulated electronically to extract the pulse envelope
(shape of the pulse without the sinusoidal oscillation). The width of the pulse envelope
is the coherence length, which determines the axial resolution of the OCT system. The
coherence length is inversely proportional with the wavelength range or bandwidth.
When the OCT system is used to image an actual tissue sample, there are many
reflections at different depths. As the reference mirror is scanned, each sample reflection
gives rise to a signal pulse when the reference delay matches it. The plot of the
demodulated interferometric signal is the axial scan waveform, which represents
amplitude of reflection vs. depth. All clinical commercial available OCT systems to date
employ SLD light sources. SLDs are similar to the diode lasers inside the common
compact disc (CD) player, but are made to emit over a wider range of wavelengths.
SLDs are used because they are economical, compact, longlasting, and emit high quality
beams that couples efficiently with an optical fiber. The resolution of clinical OCT is
basically limited by the state of SLD technology. Early retinal OCT systems typically
employ SLDs emitting around an 820nm center wavelength over a bandwidth of 20nm
full-width half-maximum (FWHM) .This limits the axial resolution to roughly 15 mm
FWHM in air and 11 mm in tissue. The Stratus OCT System has 910 mm axial
resolution in tissue.

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Figure 4.1 : Interference of waves of different wavelegths

Chapter 5
INTERFEROMETRY

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Interferometry is a family of techniques in which waves, usually electromagnetic,

are superimposed in order to extract information about the waves. Interferometry is an
important investigative technique in the fields of astronomy, fiber optics,
engineering metrology,optical metrology, oceanography, seismology, spectroscopy (and
its applications to chemistry),quantum mechanics, nuclear and particle physics, plasma
physics, remote sensing, biomolecular interactions, surface profiling, microfluidics,
mechanical stress/strain measurement, and velocimetry.
Interferometers are widely used in science and industry for the measurement of
small displacements, refractive index changes and surface irregularities. In analytical
science, interferometers are used in continuous wave Fourier transform spectroscopy to
analyze light containing features of absorption or emission associated with a substance
or mixture. Astronomical interferometer consists of two or more separate telescopes that
combine their signals, offering a resolution equivalent to that of a telescope of diameter
equal to the largest separation between its individual elements.
Interferometry makes use of the principle of superposition to combine waves in a
way that will cause the result of their combination to have some meaningful property
that is diagnostic of the original state of the waves. This works because when two waves
with the same frequency combine, the resulting pattern is determined by
the phase difference between the two waves that are in phase will undergo constructive
interference while waves that are out of phase will undergo destructive interference.
Most interferometers use light or some other form of electromagnetic wave.

Chapter 6
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MICHELSON INTERFEROMETER

A Michelson interferometer consists minimally of mirrors M1 & M2 and a beam

splitter M. In Fig 6.1, a source S emits light that hits the beam splitter (in this case, a
plate beamsplitter) surface M at point C. M is partially reflective, so part of the light is
transmitted through to point B while some is reflected in the direction of A. Both beams
recombine at point C' to produce an interference pattern incident on the detector at
point E (or on the retina of a person's eye). If there is a slight angle between the two
returning beams, for instance, then an imaging detector will record a sinusoidal fringe
pattern as shown in Fig. 6.2b. If there is perfect spatial alignment between the returning
beams, then there will not be any such pattern but rather a constant intensity over the
beam dependent on the differential pathlength; this is difficult, requiring very precise
control of the beam paths.
Fig. 6.1 shows use of a coherent (laser) source. Narrowband spectral light from
a discharge or even white light can also be used, however to obtain significant
interference contrast it is required that the differential pathlength is reduced below the
coherence length of the light source. That can be only micrometers for white light, as
discussed below.
If a lossless beamsplitter is employed, then one can show that optical energy is
conserved. At every point on the interference pattern, the power that is not directed to
the detector at E is rather present in a beam (not shown) returning in the direction of the
source.

Figure 6.1 : Path of light in Michelson Interferometer

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As seen in Fig. 6.2a and 6.2b, the observer has a direct view of mirror M1 seen
through the beam splitter, and sees a reflected image M'2 of mirror M2. The fringes can
be interpreted as the result of interference between light coming from the two virtual
images S'1 and S'2 of the original source S. The characteristics of the interference pattern
depend on the nature of the light source and the precise orientation of the mirrors and
beam splitter. In Fig. 6.2a, the optical elements are oriented so that S'1and S'2 are in line
with the observer, and the resulting interference pattern consists of circles centered on
the normal to M1and M'2 (fringes of equal inclination). If, as in Fig. 6.2b, M1 and M'2 are
tilted with respect to each other, the interference fringes will generally take the shape
of conic sections (hyperbolas), but if M1 and M'2 overlap, the fringes near the axis will
be straight, parallel, and equally spaced (fringes of equal thickness). If S is an extended
source rather than a point source as illustrated, the fringes of Fig. 6.2a must be observed
with a telescope set at infinity, while the fringes of Fig. 6.2b will be localized on the
mirrors.

Figure 6.2 : Formation of fringes in a Michelson interferometer

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Chapter 7
TYPES OF OCT
7.1:Time Domain OCT
In time domain OCT the pathlength of the reference arm is translated longitudinally in
time. A property of low coherence interferometry is that interference, i.e. the series of
dark and bright fringes, is only achieved when the path difference lies within the
coherence length of the light source. This interference is called auto correlation in a
symmetric interferometer (both arms have the same reflectivity), or cross-correlation in
the common case. The envelope of this modulation changes as pathlength difference is
varied, where the peak of the envelope corresponds to pathlength matching.
The interference of two partially coherent light beams can be expressed in terms
of the source intensity,

where

, as

represents the interferometer beam splitting ratio, and

is called the complex degree of coherence, i.e. the interference envelope and carrier
dependent on reference arm scan or time delay , and whose recovery of interest in
OCT. Due to the coherence gating effect of OCT the complex degree of coherence is
represented as a Gaussian function expressed as[16]

where
domain, and

represents the spectral width of the source in the optical frequency

is the centre optical frequency of the source. In equation (2), the

Gaussian envelope is amplitude modulated by an optical carrier. The peak of this

envelope represents the location of sample under test microstructure, with an amplitude
dependent on the reflectivity of the surface. The optical carrier is due to the Doppler

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effect resulting from scanning one arm of the interferometer, and the frequency of this
modulation is controlled by the speed of scanning. Therefore translating one arm of the
interferometer has two functions; depth scanning and a Doppler-shifted optical carrier
are accomplished by pathlength variation. In OCT, the Doppler-shifted optical carrier
has a frequency expressed as

where

is the central optical frequency of the source,

of the pathlength variation, and

is the scanning velocity

is the speed of light.

The axial and lateral resolutions of OCT are decoupled from one another; the
former being an equivalent to the coherence length of the light source and the latter
being a function of the optics. The axial resolution of OCT is defined as

7.2: Frequency Domain OCT

In frequency domain OCT the broadband interference is acquired with spectrally
separated detectors (either by encoding the optical frequency in time with a spectrally
scanning source or with a dispersive detector, like a grating and a linear detector array).
Due to the Fourier relation (Wiener-Khintchine theorem between the auto correlation
and the spectral power density) the depth scan can be immediately calculated by a
Fourier-transform from the acquired spectra, without movement of the reference
arm. This feature improves imaging speed dramatically, while the reduced losses during
a single scan improve the signal to noise proportional to the number of detection
elements. The parallel detection at multiple wavelength ranges limits the scanning
range, while the full spectral bandwidth sets the axial resolution.
7.2.1:Spatially encoded frequency domain OCT (spectral domain or Fourier
domain OCT)

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SEFD-OCT extracts spectral information by distributing different optical frequencies

onto a detector stripe (line-array CCD or CMOS) via a dispersive element . Thereby the
information of the full depth scan can be acquired within a single exposure. However,
the large signal to noise advantage of FD-OCT is reduced due to the lower dynamic
range of stripe detectors with respect to single photosensitive diodes, resulting in an
SNR (signal to noise ratio) advantage of ~10 dB at much higher speeds. This is not
much of a problem when working at 1300 nm, however, since dynamic range is not a
serious problem at this wavelength range.
The drawbacks of this technology are found in a strong fall-off of the SNR, which
is proportional to the distance from the zero delay and a sinc-type reduction of the depth
dependent sensitivity because of limited detection linewidth. (One pixel detects a quasirectangular portion of an optical frequency range instead of a single frequency, the
Fourier-transform leads to the sinc(z) behavior). Additionally the dispersive elements in
the spectroscopic detector usually do not distribute the light equally spaced in frequency
on the detector, but mostly have an inverse dependence. Therefore the signal has to be
resampled before processing, which cannot take care of the difference in local
(pixelwise) bandwidth, which results in further reduction of the signal quality. However,
the fall-off is not a serious problem with the development of new generation CCD or
photodiode array with a larger number of pixels.
Synthetic array heterodyne detection offers another approach to this problem
without the need for high dispersion.
7.2.2 : Time encoded frequency domain OCT (also swept source OCT)

TEFD-OCT tries to combine some of the advantages of standard TD and SEFD-OCT.

Here the spectral components are not encoded by spatial separation, but they are
encoded in time. The spectrum either filtered or generated in single successive
frequency steps and reconstructed before Fourier-transformation. By accommodation of
a frequency scanning light source (i.e. frequency scanning laser) the optical setup
becomes simpler than SEFD, but the problem of scanning is essentially translated from
the TD-OCT reference-arm into the TEFD-OCT light source. Here the advantage lies in
the proven high SNR detection technology, while swept laser sources achieve very
small instantaneous bandwidths (=linewidth) at very high frequencies (20200 kHz).

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Drawbacks are the nonlinearities in the wavelength (especially at high scanning

frequencies), the broadening of the linewidth at high frequencies and a high sensitivity
to movements of the scanning geometry or the sample (below the range of nanometers
within successive frequency steps).

Figure 7.2.2.1 : Time & Frequency domain OCT

Figure 7.2.2.2 :Time domain OCT setup

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Figure 7.2.2.3 : Frequency domain OCT setup

Chapter 8
COMPARISON OF TD AND FD OCT
No physical scanning of the reference mirror is required,thus FD-OCT is much faster
than TD-OCT.
The simultaneous detection of reflections from a broad range of depths is much
more efficient than TD-OCT in which signals from various depths are scanned
sequentially.
FD-OCT is fast enough to track the ulsation of blood vessels.

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Figure 8.1 : TD

OCT

Figure 8.2 : FD

OCT

Chapter 9
OCT RETINAL THICKNESS MEASUREMENT

At rst moment, it is very difcult to understand why the OCT3 software erroneously
delineates the retinal boundaries in an optimal tomogram as such exemplied in Fig. 22.
Initially, one should bear in mind that automatic retinal thickness measurements are
generated in essence by means of a mathematical calculation (algorithm). The
algorithm identies differences in the image reectance patterns in each A-scan (up to
512 A-scans in OCT 3) that compose one tomogram (B-scan), and assumes that the
distance between two relatively high reective structures represents the retinal thickness
at that A-scan. As a result, the OCT software locates the presumed inner retina boundary
at the vitreoretinal interface (rst high reective structure) and the presumed outer retina
boundary at the retinal pigment epithelial-photoreceptor outer segment interface (second
high reective structure).
The algorithm also compares the shape of one A-scan to adjacent A-scans, once
great differences in shape are not expected to occur in side-by-side A-scans. The
software then places a line on the inner vitreoretinal interface and another on the retinal

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pigment epithelium (RPE)-outer retinal interface and determines retinal thickness as the
distance between these lines at each measurement point along the scans x-axis.
Therefore, even in ne-looking B-scans, errors in retinal boundaries delineation may
occur.

Figure 9.1 : OCT thickness measurement

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Chapter 10
SCANNING SCHEMES

Focusing the light beam to a point on the surface of the sample under test, and
recombining the reflected light with the reference will yield an interferogram with
sample information corresponding to a single A-scan (Z axis only). Scanning of the
sample can be accomplished by either scanning the light on the sample, or by moving
the sample under test. A linear scan will yield a two-dimensional data set corresponding
to a cross-sectional image (X-Z axes scan), whereas an area scan achieves a threedimensional data set corresponding to a volumetric image (X-Y-Z axes scan), also
called full-field OCT.
10.1 : Single Point (Confocal) OCT
Systems based on single point, or flying-spot time domain OCT, must scan the sample
in two lateral dimensions and reconstruct a three-dimensional image using depth
information obtained by coherence-gating through an axially scanning reference arm .
10.2 : Parallel (or full field) OCT
Parallel OCT using a charge-coupled device (CCD) camera has been used in which the
sample is full-field illuminated and en face imaged with the CCD, hence eliminating the
electromechanical lateral scan. By stepping the reference mirror and recording
successive en face images a three-dimensional representation can be reconstructed.
Three-dimensional OCT using a CCD camera was demonstrated in a phase-stepped
technique, using geometric phase shifting with a Linnik interferometer, utilising a pair
of CCDs and heterodyne detection, and in a Linnik interferometer with an oscillating
reference mirror and axial translation stage.[Central to the CCD approach is the

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necessity for either very fast CCDs or carrier generation separate to the stepping
reference mirror to track the high frequency OCT carrier.
10.3 : Smart Detector Array For Parallel TD-OCT

A two-dimensional smart detector array, fabricated using a 2 m complementary metaloxide-semiconductor (CMOS) process, was used to demonstrate full-field OCT.

Chapter 11
REGIONS OF RETINA IN A OCT

The vertebrate retina has ten distinct layers. From closest to farthest from the vitreous
body - that is, from closest to the front exterior of the head towards the interior and back
of the head:
1. Inner limiting membrane basement membrane elaborated by Mller cells
2. Nerve fibre layer axons of the ganglion cell nuclei (note that a thin layer of
Mller cell footplates exists between this layer and the inner limiting
membrane)
3. Ganglion cell layer contains nuclei of ganglion cells, the axons of which
become the optic nerve fibres for messages and some displaced amacrine cells
4. Inner plexiform layer contains the synapse between the bipolar cell axons and
the dendrites of the ganglion and amacrine cells.
5. Inner nuclear layer contains the nuclei and surrounding cell bodies (perikarya)
of the amacrine cells, bipolar cells and horizontal cells.
6. Outer plexiform layer projections of rods and cones ending in the rod spherule
and cone pedicle, respectively. These make synapses with dendrites of bipolar
cells.[1] In the macular region, this is known as the Fiber layer of Henle.

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7. Outer nuclear layer cell bodies of rods and cones

8. External limiting membrane layer that separates the inner segment portions of
the photoreceptors from their cell nucleus
9. Layer of rods and cones layer of rod cells and cone cells
10.Retinal pigment epithelium - single layer of cuboidal cells (with extrusions not
shown in diagram). This is closest to the choroid.

Figure 11.1 : Retina Layers in a OCT

Chapter 12
OCT SCANS

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Figure 12.1 : OCT Scans

A macular hole is a small break in the macula, located in the center of the eye's
light-sensitive tissue called the retina. The macula provides the sharp, central vision we
need for reading, driving, and seeing fine detail. A macular hole can cause blurred and
distorted central vision.

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Vitelliform macular dystrophy causes a fatty yellow pigment (lipofuscin) to build

up in cells underlying the macula. Over time, the abnormal accumulation of this
substance can damage cells that are critical for clear central vision.
Stargardt disease, or fundus flavimaculatus, is an inherited form of
juvenile macular degeneration that causes progressive visioion loss usually to the point
of legal blindness.
central serous chorioretinopathy (CSC), is an eye disease which causes visual
impairment, often temporary, usually in one eye.
The OCT can be used to identify these retinal diseases.
12.1 : OTHER APPLICATIONS
Optical coherence tomography is an established medical imaging technique. It is widely
used, for example, to obtain high-resolution images of the anterior segment of
the eyeand the retina, which can, for example, provide a straightforward method of
assessing axonal integrity in multiple sclerosis, as well as macular degeneration.
Research indicates that OCT may be a reliable tool for monitoring the progression
of glaucoma. Researchers also seek to develop a method that uses frequency domain
OCT to imagecoronary arteries in order to detect vulnerable lipid-rich plaques.
Researchers have used OCT to produce detailed images of mice brains, through a
"window" made of zirconia that has been modified to be transparent and implanted in
the skull.
Optical coherence tomography is also applicable and increasingly used
in industrial applications, such as nondestructive testing(NDT), material thickness
measurements, and in particular thin silicon wafers, and compound semiconductor
wafers thickness measurements,, surface roughness characterization, surface and crosssection imaging, and volume loss measurements. OCT systems with feedback can be
used to control manufacturing processes. With high speed data acquisition, and submicron resolution, OCT is adaptable to perform both inline and off-line. Due to the high
volume of produced pills, an interesting field of application is in the pharmaceutical
industry to control the coating of tablets. Fiber-based OCT systems are particularly
adaptable to industrial environments. These can access and scan interiors of hard-toreach spaces, and are able to operate in hostile environments - whether radioactive,

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cryogenic, or very hot. Novel optical biomedical diagnostic and imaging technologies
are currently being developed to solve problems in biology and medicine. As of 2014,
attempts have been made to use optical coherence tomography to identify root canals in
teeth, specifically canal in the maxillary molar, however, there's no difference with the
current methods of dental operatory microscope.

Figure 12.2 : OCT of esophagus

Chapter 13
OCT SYSTEM
The OCT system employed in this study was specifically designed to feature an axial
resolution in the range of few micrometers in order to facilitate imaging of the small
structures of the RPE layer while providing an A-Scan rate suitable for in-vivo

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measurements. Because of the 2c=g dependence of the axial resolution, a system with
center wavelength c 830 nm was realized using a broadband superluminescent lightemitting diode (SLED) light source combining four SLED modules (EBS8C10, Exalos
AG, Switzerland), resulting in a 3 dB bandwidth of 170 nm and an axial resolution
of 1.78 m in air, which was determined by the intensity-based full width half maximum
of the absolute value of the light source Fourier transform. The in-house developed
spectrometer consists of a volume phase holographic dispersion grating with 1200
lp/mm, an optical system using spherical 2-inch optics, a custom made field flattening
lens, and a 2048-pixel line scan camera (AVIIVA EM4, e2v, U.K.) with 70 kHz line rate.
The OCT system was combined with the laser treatment system using a
dichroic mirror . The sample beam was focused onto the samples using a standard 2D
galvo scanner unit and a SDO (Wild MedTec, Wien, Austria) with a retinal spot size of
approximately 35 m for in-vivo and time-resolved measurements. For measurements on
porcine eyes with removed anterior segments, an achromatic focusing lens with f33
mm (retinal spot size approx. 13 m) was employed. Spot sizes were measured and
confirmed using a beam profiler (WinCamD-UHR, DataRay Inc., Bella Vista, CA,
USA). For the experiments in this paper, the incident power of the OCT probing beam
at the sample arm was set to 1.9 mW and reference arm power was adjusted using a
simple neutral density damping element. Integration time of the OCT scans was set to a
minimum value of 14 s to achieve the highest frame rate. The sample focus position was
controlled and optimized for each recording by simultaneously moving the delivery
optics and the reference arm until signal quality and SNR reached a maximum. OCT
volume data were recorded with two dimensional telecentric line scanning using the
scanning unit. To correct the large dispersion mismatch of the OCT system caused by
the multiple lenses in the sample arm and intensified by the broad bandwidth of the light
source, dispersion compensation glass (26 mm N-BK7) was added to the reference arm.
Residual and higher order dispersion was compensated for numerically during postprocessing of the OCT data. With the described system, a maximum sensitivity of 102
dB with an integration time of 14 s was achieved with a fall-off of approximately 5 dB
over the first mm and approximately 25 dB over the measurement range of 2.3 mm.
OCT data acquisition and post-processing was implemented in a proprietary
LabVIEW framework (NI, Austin, Texas, USA). Averaging of OCT scans was enabled
by the software but resulted in a reduced acquisition speed. Averaging was thus limited
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to a value of four consecutive scans without using frame-to-frame registration because

of the static setup. Processed data were stretched and re-sampled frame by frame to
correct for the difference in lateral and axial resolution.
Sample Preparation Porcine eyes were collected from a local slaughterhouse
where sacrificing of the animal took place 14 hours before the experiments. Upon
collection, the porcine eyes were stored on ice
Schematic setup of the FD-OCT system at 830 nm coupled to the laser
photocoagulation therapy setup. The OCT setup includes light source, fiber coupler,
dichroic mirror, and spectrometer, and the therapy system includes the treatment laser
source and the scanning digital ophthalmoscope (SDO). OCT light source spectrum and
PSF are shown in the insets on the top left. The box on the bottom right shows the
alternative optical setup used if anterior segments were removed and covered in DMEM
solution (Gibco Life Technologies, Carlsbad, CA, USA) to prevent rapid degradation.
Before exposure to the laser energy, the anterior segment including cornea and lens was
carefully removed by circumbulbar cutting the eyes posterior to the limbus to prevent
opacity effects of the cornea to have influence on the experiments. The vitreous was
kept in place and covered with a round d20 mm microscope cover slip. The porcine
eyes were fixated in a specifically designed sample chamber and subsequently treated
with the treatment laser.
Treatment Laser- For the laser treatment, a prototype laser emitting at 577 nm
(Merilas, Meridian AG, Thun, Switzerland) was used. The laser was operated in a quasicontinuous mode with pulse repetition frequency selectable between 15 kHz and 50
kHz, which, in all cases, exceeded the value for pulse effects in the tissue . Therefore,
treatment effects and temperature distributions are similar or identical to cw irradiation.
Threshold energy considerations in this paper are thus referenced to published values of
cw (single pulse) laser coagulation. The treatment laser was coupled into the setup using
a standard 50 m fiber with NA0:22 and guided onto the retina using a f35 mm
achromatic collimating lens, a dichroic mirror and a fixed protected silver mirror. The
collimated treatment laser was directly focused onto the porcine retina using a f120
mm focus lens. The optical setup featured a treatment laser spot size of approximately
130 m on the retina. The retinal spot size was confirmed by microscopic inspection of

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the lesions. Lesions were placed using the stage of the SDO and the video feedback
provided by the SDO camera chip. The laser lesions were set with trains of pulses
adding up to a total irradiation time of 10 ms. Total applied energies varied from 0.7 to
20 mJ, which is equivalent to laser powers of between 70 mW and 2 W.
Histology - In this study, histological sections of the laser lesions were prepared to
compare the structural changes to the alterations of optical properties detected with
OCT as histology can be used to directly asses tissue damage caused by laser irradiation
. After laser treatment, the enucleated porcine eyes were fixated in Davidson's solution
mixed according to for 24 h before the samples were washed in a flush mixture (60%
ethanol, 40% tap water). The samples were then trimmed to an approximate size of 20
mm 10 mm using high energy marker lesions as a reference and prepared for
embedding by paraffinization. Samples were embedded in paraffin and cut using a
microtome. Sections were taken every 30 m and stained with H&E staining.
Histological section were imaged with a transmitted-light microscope (AxioPlan 2, Carl
Zeiss AG, Germany) with 20 magnification. For the purpose of matching the
histological images with the OCT data, full tile scan images (2.5 mm 15 mm) of the
histological sections were acquired using the motorized stage of the microscope and the
ImageJ stitching tool. The acquired single tiles featured a subsection of 698 m 552 m
with a lateral resolution of 536 nm in both directions. Tiles were acquired with a 7%
overlap to enable stitching.
Visual Inspection Immediately after exposure to the laser energy, images of
the lesion patterns were taken using a digital microscope (Reflecta GmbH, Rottenburg,
Germany) with a magnification of 40x. The retinal lesions were classified manually into
four classes by two independent observers. The Strong Visible (SV) class includes
lesions creating a clearly visible spot on the retina and a visible distortion of the layers.
This lesion class shows affected tissue within an area larger than the spot size of the
treatment laser and tended to further increase in size for 1030 minutes after laser
application. The Visible (V) class includes lesions that are still clearly visible but did
not show any layer distortion or size increase after laser application. The Barely
Visible (BV) and the Invisible (IV) class include the lesions that were barely visible
as grayish spots and the lesions that could not be detected ophthalmoscopically,
respectively.

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Chapter 13
CONCLUSION
The introduction of OCT in ophthalmology represents a definitive change in the way
doctors understand and treat several diseases affecting the retina. It is quite likely as
well, that the role of OCT as a method to diagnose and manage glaucoma will be further
defined in the near future. Understanding of the basic principles in which OCT relays on
is essential to understand its actual limitations and to use this technology with wisdom.
We have already learned a lot with data provided by first generations of OCT, and there
is much more to learn with forthcoming data from numerous ongoing studies worldwide
that, presently, are using third generation OCT systems. A huge leap forward in

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improving OCT imaging performance is expected to occur within the next few years
with the commercial availability of next generation OCT.

Chapter 14
QUESTIONS AND ANSWERS
1) Which is the most used and best interferometry technique available?
Ans) The Mach-Zehnder interferometer's relatively large and freely accessible working
space, and its flexibility in locating the fringes has made it the interferometer of choice
for visualising flow in wind tunnels, and for flow visualization studies in general. It is
frequently used in the fields of aerodynamics, plasma physics and heat transfer to
measure pressure, density, and temperature changes in gases.
2) Can TD-OCT be used where FD-OCT is used?

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Ans) TD-OCT acquires data sequentially,while FD-OCT acquires data simultaneously

making it able to obtain numerous number of data from various depths of retina at a
time.This makes FD-OCT faster and makes it produce a more clearer image than TDOCT.SO TD-OCT can be used where FD-OCT is used,but would be less efficient in
comparison with former.
3) Tissues can be in the range of nanometer scale,so can we apply OCT to view such
tissues?
Ans)As per the experiments conducted the tissues found in the retina come in the range
of 10 to 14 micrometer and they were able to obtain a axial resolution of 1.48
micrometer ,whereas standard OCT systems have obtained only upto 6 micrometer
resolution.
4) What do you mean by false colors?
Ans) A false color image sacrifices natural color understanding in order to ease the
detection of features that are not readily discernible.So each type of region would be
signified by a color and this color is not the original color of the specimen ,but carries a
meaning.In OCT it is used to denote different frequency levels.

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