fish eye imaging delhi.pdf

High-resolution corneal topography and tomography of fish eye using wide-field white light interference microscopyVishal 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 View Table of Contents: http://apl.aip.org/resource/1/APPLAB/v102/i15 Published by the AIP Publishing LLC.

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High-resolution corneal topography and tomography of fisheye using wide-field white light interference microscopy

Vishal Srivastava,1,a) Sreyankar Nandy,2 and Dalip Singh Mehta2,b)1Laser Applications and Holography Laboratory, Instrument Design Development Centre,Indian Institute of Technology Delhi, Hauz Khas, New Delhi-110016, India2Department 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

recorded data is improved.VC 2013 AIP Publishing LLC [http://dx.doi.org/10.1063/1.4802084]

Optical coherence tomography (OCT) is a non-contact,

non-invasive, and non-destructive technique for high resolu-

tion 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 possi-

bility of three dimensional imaging as well as imaging speed

has been greatly increased by the introduction of Fourier-

domain OCT in 2002 from axial resolution (36 lm) tospeed (20 000-50 000 lines/s).513 The cornea is a clear mem-

brane 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 re-

fractive 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 theconventional OCT systems require lateral mechanical scan-

ning, therefore, OCT has been further extended to parallel

and full-field detection mode.1922 In Full-field OCT (FF-

OCT), 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 coher-

ence tomographic system has an advantage of high axial

resolution as well as high transverse resolution as compared

to cross-sectional OCT techniques.2326 The FF-OCT sys-

tems 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 ampli-

tude 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. Low-

coherence white light is divided into two beams by a beam

splitter (BS) and propagates into the sample and the refer-

ence 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 I0x; y1 Vczcosf/x; y; zg; (1)

where I0(x, y) is the background intensity, V is fringe con-

trast, cz exp 2p zz0lc 2

the coherence envelope of

low coherence light source with lc 0.44 k02/Dk being thecoherence length, k0 is the central wavelength, z is the verti-cal scanning position of PZT scanner, z0 is the peak position

of coherence envelope, / (x, y, z) is the phase map. The cen-tral wavelength k0 is the wavelength where the phase differ-ence 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,

a)[email protected])[email protected]

0003-6951/2013/102(15)/153701/5/$30.00 VC 2013 AIP Publishing LLC102, 153701-1

APPLIED PHYSICS LETTERS 102, 153701 (2013)


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 shiftedinterferograms at each depth. Phase map for every interfero-

gram 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

/ix; y tan12fI2x; y I4x; yg

I1x; y 2I3x; y I5x; y

: (2)

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

wavelength, e.g., k0 560 nm for which the phase step isexactly p/2. Equation (2) provides the so-called modulo 2pphase at each pixel, whose value ranges from 0 to 2p.Minimum LP-norm two-dimensional phase unwrapping algo-

rithm 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 2pk0

nsamplex; y nmediumx; yZ; (3)

where nsample (x, y) and nmedium (x, y) are the refractive indi-

ces of sample and medium, respectively, and Z is the depth,

nsamplex; y k02pZ

D/x; y nmediumx; y: (4)

The schematic diagram of the experimental system is shown

in Fig. 1. More details can be found in our previous publica-

tion,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 FF-

WL-OCT system were determined experimentally. The axial

resolution of the system was measured with the same inter-

ferometric microscope using 20 Mirau objective. In placeof 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 meas-

ured directly. This comes out to be 1.6 lm as shown in Fig.2(a). Hence axial resolution is 0.8 lm. To determine thetransverse resolution of present system, a 1 lm diameterpolystyrene 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 Mirauobjective lens. Hence the PSF which is basically a line pro-

file 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 reso-

lution measured was 1.3 lm. The axial and transverse resolu-tions obtained for the present system are 0.8 lm and 1.3 lm,respectively, which is a high resolution as compare to con-

ventional fiber based OCT systems. The area of illumination

on the object is 2 2 mm2 and thus does not require any Bscan mechanism and hence is wide-field.

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

system.

153701-2 Srivastava, Nandy, and Mehta Appl. Phys. Lett. 102, 153701 (2013)


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 producedby the PZT phase shifter at a wavelength of 560 nm in the

present case. We have taken series of interferograms at dif-

ferent depths of cornea of the eye by moving the transla-

tional stage in the Z-direction in steps with the help of PZT.

The depth of penetration was approximately 40 lm. Thetheoretical axial and spatial resolution of the system is

0.8 lm and 1.3 lm which may degrade as it travels throughtissues. Our system produces topographic images in the x-y

(en face) orientation. The sample is moved step by step inthe axial direction to acquire a stack of topographic images,

forming a 3D data set (tomographic image). Figure 3(a)

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

transverse resolution of FF-WL-OCT

system.

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



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 interfero-

gram 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 1lm were analyzed to reconstruct thephase 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 usefulfor finding the corneal power. The RI of the epithelium layer

at Z 5lm, is 1.388 (nmedium 1). In the case of closedfringe 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 wellas cross-sectional images which provide complementary in-

formation. 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 cor-

neal imaging. FF-WL-OCT system allows high-resolution

imaging of fish cornea. The results shown are of high resolu-

tion that demonstrates the strength of this technique in imag-

ing unstained fish eye. These topographic images may be

helpful in finding the corneal disease and detecting the irreg-

ularities 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 sta-

tionary. For higher penetration, we have to image each layer

by removing previous layer. For human eye, some modifica-

tion 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 1.3lm) than conventional super-luminence 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.

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



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

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