High-resolution corneal topography and tomography of fish eye using widefield white light interference microscopy

Vishal Srivastava, Sreyankar Nandy, and Dalip Singh Mehta

Citation: Appl. Phys. Lett. 102, 153701 (2013); doi: 10.1063/1.4802084
View online: http://dx.doi.org/10.1063/1.4802084
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APPLIED PHYSICS LETTERS 102, 153701 (2013)

High-resolution corneal topography and tomography of fish

eye using wide-field white light interference microscopy
Vishal Srivastava,1,a) Sreyankar Nandy,2 and Dalip Singh Mehta2,b)
1

Laser Applications and Holography Laboratory, Instrument Design Development Centre,

Indian Institute of Technology Delhi, Hauz Khas, New Delhi-110016, India
2
Department of Physics, Indian Institute of Technology Delhi, Hauz Khas, New Delhi-110016, India

(Received 9 February 2013; accepted 2 April 2013; published online 18 April 2013)
Topography and tomography of fish cornea is reconstructed using high resolution white light
interference microscopy. White light interferograms at different depths were recorded by moving the
object axially. For each depth position, five phase shifted interferograms were recorded and analyzed.
From the reconstructed phase maps, the corneal topography and hence the refractive index was
determined and from amplitude images the cross-sectional image of fish cornea was reconstructed. In
the present method, we utilize a nearly common-path interference microscope and wide field
illumination and hence do not require any mechanical B-scan. Therefore, the phase stability of the
C 2013 AIP Publishing LLC [http://dx.doi.org/10.1063/1.4802084]
recorded data is improved. V
Optical coherence tomography (OCT) is a non-contact,
non-invasive, and non-destructive technique for high resolution tomographic imaging of microstructures in biological
tissues. The most important application of the OCT is in the
field of ophthalmologic investigations of the health of cornea
and the retina.14 OCT uses low-coherence interferometry to
produce a two-dimensional image of optically back scattered
light from internal tissue microstructures. Significant
research and development have taken place by using various
OCT techniques for corneal and retinal imaging. The possibility of three dimensional imaging as well as imaging speed
has been greatly increased by the introduction of Fourierdomain OCT in 2002 from axial resolution (36 lm) to
speed (20 000-50 000 lines/s).513 The cornea is a clear membrane that covers the front of the eye and plays an important
role in focusing images on the retina. About 70% of total
dioptic power is because of the cornea which is the major refractive surface of the eye.14 Hence small changes in the
shape of the cornea will affect clarity and required treatment
or surgery. Corneal topography gives both qualitative and
quantitative information which is helpful in finding the
health of cornea. To optimize image quality, the choice of
light source is critical. For improved axial resolution, a broad
band light source is required whose coherence length is very
small. Hence interference will only occur when optical path
length is within the coherence length of the light source and
precise slice selection in the sample is therefore possible.
White light source is an ideal source for high axial resolution
because of its broad band width. White light is already used
for ex-vivo imaging of biological tissues.1518 Most of the
conventional OCT systems require lateral mechanical scanning, therefore, OCT has been further extended to parallel
and full-field detection mode.1922 In Full-field OCT (FFOCT), lateral mechanical scanning is not required and with
the help of high numerical aperture objective lenses, one can
obtain high transverse resolution. Full-field optical coherence tomographic system has an advantage of high axial
a)

vishalsrivastava17@gmail.com
mehtads@physics.iitd.ac.in

b)

0003-6951/2013/102(15)/153701/5/$30.00

resolution as well as high transverse resolution as compared

to cross-sectional OCT techniques.2326 The FF-OCT systems have recently become more popular, in which the depth
and lateral scan are simultaneously obtained. But most of the
conventional FF-OCT systems provide only amplitude
images and phase information is not recovered. In this paper,
we report the topography of fish eye cornea using wide-field
white light optical coherence tomographic (WF-WL-OCT)
system with a coaxial and common path optical system based
on Mirau interferometric objective lens. Simultaneous amplitude and phase images were obtained by means of recording
2D spatial interferograms at different depths. Multiple phase
shifted interferograms were recorded with the help of a
piezo-electric transducer (PZT), and both the amplitude and
the phase map of the interference fringe signal were
reconstructed.
The schematic diagram of a FF-WL-OCT system based
on a compact Mirau interferometer is shown in Fig. 1. Lowcoherence white light is divided into two beams by a beam
splitter (BS) and propagates into the sample and the reference arms of a Mirau interferometer. The reflected light
from the reference mirror and backscattered light from the
sample interferes at the beam splitter and is then detected by
the charge-coupled device (CCD) camera and the intensity
distribution of a white light interferogram recorded by an
area detector can be written as27
Ix; y; z I0 x; y1 Vczcosf/x; y; zg;

(1)

intensity, V is fringe conwhere I0(x, y) is the background

 2 
zz0
the coherence envelope of
trast, cz exp 2p lc
low coherence light source with lc 0.44 k02/Dk being the
coherence length, k0 is the central wavelength, z is the vertical scanning position of PZT scanner, z0 is the peak position
of coherence envelope, / (x, y, z) is the phase map. The central wavelength k0 is the wavelength where the phase difference between the red, green and blue part of the white-light
interference fringe becomes equal to zero. There are various
techniques to find out the central wavelength, such as,

102, 153701-1

C 2013 AIP Publishing LLC

V

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153701-2

Srivastava, Nandy, and Mehta

Appl. Phys. Lett. 102, 153701 (2013)

FIG. 1. The schematic diagram of a FF-WL-OCT

system.

phase-shift, frequency domain analysis, coherent correlation

weight-center, etc.8,9 We used phase crossing algorithm to
indentify the central wavelength28 which is 560 nm in the
present case and lc 1.6 lm. We recorded five phase shifted
interferograms at each depth. Phase map for every interferogram was computed using Eq. (2) given below. A 5-step
phase shifting algorithm is used to reconstruct the phase map
given by the following expression,27
/i x; y tan

1


2fI2 x; y  I4 x; yg
:
I1 x; y  2I3 x; y I5 x; y

(2)

Equation (2) is used to extract the phase map for central

wavelength, e.g., k0 560 nm for which the phase step is
exactly p/2. Equation (2) provides the so-called modulo 2p
phase at each pixel, whose value ranges from 0 to 2p.
Minimum LP-norm two-dimensional phase unwrapping algorithm is used to obtain the continuous phase map.29 The
reconstructed phase maps can be utilized to determine the
refractive index of cornea at central wavelength using the
following expression:
D/x; y

2p
nsample x; y  nmedium x; yZ;
k0

(3)

where nsample (x, y) and nmedium (x, y) are the refractive indices of sample and medium, respectively, and Z is the depth,
nsample x; y

k0
 D/x; y nmedium x; y:
2pZ

(4)

The schematic diagram of the experimental system is shown

in Fig. 1. More details can be found in our previous publication,30 however, the system is briefly described here. Light
emitted from the white light source is collimated by a lens
and made incident onto a beam splitter, which is directed
towards the Mirau interferometeric objective lens (Model
No. 403115, 20/0.40 DI, WD 4.7 Nikon, Japan) to image

the anterior segment of the fish eye. This objective lens is

attached with a PZT (Peizo, Jena, MIPOS 3). The PZT is
driven by an amplifier which is controlled by a personal
computer and it moves the inbuilt reference mirror in the
vertical direction for introducing the phase shifts between
the reference and object beams. The phase shifted white light
interferograms are recorded by a single-chip colour CCD
camera (Artray, IP 150M, Sony ICX 205AK), 10FPS, with
total pixels 1024 (H)  1360 (V) and pixel size 6.25 lm.
Aiming laser is used to ensure light incident on the cornea.
To record the interference at different depths in the eye, we
vary the height of the translation stage in the Z-direction and
tuned to make them focused at different depths in the eye.
Interferometric images were captured by the CCD camera
and were transferred to a computer for signal processing and
image display. The axial and transverse resolutions of FFWL-OCT system were determined experimentally. The axial
resolution of the system was measured with the same interferometric microscope using 20 Mirau objective. In place
of sample, we placed a reflective mirror and whose position
was scanned axially (along Z-direction) with the same
microscope such that the coherence function could be measured directly. This comes out to be 1.6 lm as shown in Fig.
2(a). Hence axial resolution is 0.8 lm. To determine the
transverse resolution of present system, a 1 lm diameter
polystyrene beads were imaged. The image of each bead in
the CCD camera represents the point-spread function (PSF)
of the microscope which is measured by the 20 Mirau
objective lens. Hence the PSF which is basically a line profile as a function of lateral position is shown in Fig. 2(b)
(where Y-axis is in normalized intensity (A.U.)) from which,
the full width half maximum (FWHM), i.e., transverse resolution measured was 1.3 lm. The axial and transverse resolutions obtained for the present system are 0.8 lm and 1.3 lm,
respectively, which is a high resolution as compare to conventional fiber based OCT systems. The area of illumination
on the object is 2  2 mm2 and thus does not require any B
scan mechanism and hence is wide-field.

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153701-3

Srivastava, Nandy, and Mehta

Appl. Phys. Lett. 102, 153701 (2013)

FIG. 2. (a) Axial resolution and (b)

transverse resolution of FF-WL-OCT
system.

To demonstrate the capability of the white light OCT

system, we tested it on the fish cornea. Fish eye cornea is
very similar to human eye cornea and generally consists of
five layers such as, epithelium, Bowmans membrane,
stroma, Descemets membrane, and endothelium. In our
experiment, we took small Indian mourala fish obtained
from a local vendor. The fresh fish eye was placed on a
microscope object stage (X-Y-Z translation stage). In order
to acquire a two-dimensional image of the fish eye it was put
on a linear translation stage. By means of adjusting the stage,
both imaging and interference fringes were produced. Five
phase shifted interferograms were recorded of the dispersed

fringe pattern, corresponding to phase steps of 90 produced

by the PZT phase shifter at a wavelength of 560 nm in the
present case. We have taken series of interferograms at different depths of cornea of the eye by moving the translational stage in the Z-direction in steps with the help of PZT.
The depth of penetration was approximately 40 lm. The
theoretical axial and spatial resolution of the system is
0.8 lm and 1.3 lm which may degrade as it travels through
tissues. Our system produces topographic images in the x-y
(en face) orientation. The sample is moved step by step in
the axial direction to acquire a stack of topographic images,
forming a 3D data set (tomographic image). Figure 3(a)

FIG. 3. (a) White light interferograms and (b) wrapped phase maps at 10 lm, 20 lm, and 38 lm, respectively, along axial direction.

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153701-4

Srivastava, Nandy, and Mehta

Appl. Phys. Lett. 102, 153701 (2013)

FIG. 4. (a) Unwrapped phase maps at 10 lm, 20 lm, and 38 lm, respectively, along axial direction (b) 3D unwrapped phase map at 40 lm along axial direction
and (c) ex vivo cross-sectional and en face image of epithelium layer, Bowmans and Stroma layer of fish cornea.

shows the recorded white light interferogram at different

depths of the cornea of the eye along the Z-direction. The
visibility is maximum for zero optical path difference (OPD)
and decreases as the OPD increases and becomes zero when
optical path difference is greater than the coherence length.
The phase was calculated using Eq. (2). Figure 3(b) shows
the wrapped phase map of the recorded white light interferogram at different depths of the cornea of fish eye. It is shown
in the Fig. 3(b) that as we increase the depth, the fringe area
as well as the fringe density (due to the curvature of the eye)
has been increased. Five phase-shifted interferograms at
each depth of step size 1 lm were analyzed to reconstruct the
phase map. These values of the phases were unwrapped.
Figure 4(a) shows the unwrapped phase map of the cornea at
different depths along Z-direction. Figure 4(b) shows the 3D
phase image of the cornea in axial direction at Z 40 lm.
From this phase map, we can find out the refractive index
(RI) using Eq. (4), where k0 560 nm which may be useful
for finding the corneal power. The RI of the epithelium layer
at Z 5 lm, is 1.388 (nmedium 1). In the case of closed
fringe pattern or in circular interference, we cannot find out
the OCT image of cornea by Fourier transform or phase
shifting method directly because the Fourier spectra of the
interferogram are not separated completely. For finding the
OCT image of cornea, we used coordinate transformation
technique.31 From 3D data set, we obtain the en face as well
as cross-sectional images which provide complementary information. The information that is not seen in en face (x, y)
image can be revealed from the cross section images and

vice versa. Figure 4(c) is an amplitude image which, shows

the cross-sectional and en face images of epithelial,
Bowmans layer and stroma of the cornea of fish eye.32 In
Fig. 4(c), the microstructures of the cornea epithelium layers
cells and stroma are clearly visualized. The quality of the
image is as good as other OCT technology dedicated to corneal imaging. FF-WL-OCT system allows high-resolution
imaging of fish cornea. The results shown are of high resolution that demonstrates the strength of this technique in imaging unstained fish eye. These topographic images may be
helpful in finding the corneal disease and detecting the irregularities in the cornea. Though this system has high spatial
resolution but the main disadvantages of the present system
are that its depth of penetration is low and object must be stationary. For higher penetration, we have to image each layer
by removing previous layer. For human eye, some modification in the set-up are required.
In this paper, we have demonstrated the applicability of the
FF-WL-OCT system for quantitative analysis of the cornea of
fish. Our system provides much higher spatial resolution
(axial  transverse, 0.8 lm  1.3 lm) than conventional superluminence diode based OCT systems. RI has been calculated
from the phase map which can be useful in finding the corneal
power. However, the most important advantage is that
topographic analysis can be done along with the high-quality
cross-sectional imaging at a very low price and great ease of
use compare to the other conventional OCT systems. Thus,
FF-WL-OCT may be helpful in detecting the disease at a very
early stage.

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153701-5

Srivastava, Nandy, and Mehta

Financial assistance from DST Delhi, Govt. of India for the

project No. SR/S2/LOP-0021/2008 is gratefully acknowledged.
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