We describe the development of a single-mode fibre based optical coherence tomography set-up and its use for non-invasive optical imaging of adult zebrafish (Danio rerio) eyes. The free-space axial and lateral resolutions of the set-up were estimated to be ~ 11 and 17 μm respectively. Images of whole eye, cornea and retina acquired with the set-up have been used to estimate several ocular parameters, viz. corneal thickness, mean retinal thickness and effective refractive index of the crystalline lens

Divakar Rao, K.

Gupta, P. K.

Patel, H. S.

Verma, Y.

English

Publications of the IAS Fellows

RESEARCH COMMUNICATIONS  
 
CURRENT SCIENCE, VOL. 90, NO. 11, 10 JUNE 2006 1506 
*For correspondence. (e-mail: kdivakar@cat.ernet.in) 
Non-invasive ophthalmic imaging of 
adult zebrafish eye using optical  
coherence tomography 
 
K. Divakar Rao*, Y. Verma, H. S. Patel and  
P. K. Gupta 
Biomedical Applications Section, Raja Ramanna Centre for Advanced  
Technology, Indore 452 013, India 
 
We describe the development of a single-mode fibre 
based optical coherence tomography set-up and its use 
for non-invasive optical imaging of adult zebrafish 
(Danio rerio) eyes. The free-space axial and lateral 
resolutions of the set-up were estimated to be ~ 11 and 
17 mm respectively. Images of whole eye, cornea and 
retina acquired with the set-up have been used to esti-
mate several ocular parameters, viz. corneal thickness, 
mean retinal thickness and effective refractive index 
of the crystalline lens.  
 
Keywords: Eyes, non-invasive ophthalmic imaging, opti-
cal coherence tomography, zebrafish. 
 
USE of optical techniques for biomedical imaging is a 
topic of considerable current interest. This is motivated by 
the potential of optical techniques to provide sub-millimetre 
resolution imaging without the need for ionizing rad ation 
and associated risks1. The fundamental problem with opti-
cal imaging is that in contrast to X-rays, optical photons 
are strongly scattered in the tissue, which leads to blur-
ring of the image. Sveral approaches can be used to pick 
out the useful image-bearing light from the background 
multiply scattered light2. These exploit the depolarization 
or loss of coherence of scattered light or the fact that scat-
tered light emerges from the tissu in all directions and 
also takes a longer time to emerge compared to the unscat-
tered (ballistic) or predominantly forward-scattered (snake-
like) components. The latter essentially travels in the 
forward direction and so arrives earlier. Coherence gating 
filters-out ballistic photons with the highest image infor-
mation and hence can provide images with high resolu-
tion. Optical coherence tomography (OCT), the approach 
that exploits coherence gating for optical imaging has 
emerged as a rapid, non-contact and noninvasive, high-
resolution imaging technique and is finding clinical applica-
tions in ophthalmology, dermatology, etc.3,4. The two es-
sential components of an OCT set-up are a low coherence 
light source and a Michelson interferometer set-up, one 
arm of which has the sample and the other arm a refer-
ence mirror. Light reflected from a layer of the sample 
and the reference mirror will interfere when the two path 
lengths are within the coherence length of the source, 
which is typically less than 10 mm. Axial scanning of the 
reference mirror helps record interferograms from differ-
ent depths of the sample. Further, scanning of the refer-
ence mirror at constant speed results in Doppler shift of 
the reference signal. Interference of th  Doppler-shifted 
reference signal with that reflected from a specific depth 
in the sample (such that the optical path length of the two 
interferometer arms are within coherence length) results 
in interference signal at Doppler frequency, which can be 
isolated from the rest of the backscattered signal by a 
suitable bandpass filter or lock-in detection system. Two-
dimensional cross-sectional images and three-dimensional 
tomograms of the backscattered intensity distribution 
within the sample can be obtained by recording the inter-
ference signals from various axial and transverse posi-
tions on the sample. Two-dimensional cross-sectional 
images are constructed by performing measurements of 
the echo time delay of light at different transverse positions 
by scanning either the light beam or the sample. 
 Here we report the implementation of single-mode fibre-
based OCT set-up and demonstrate its use for imaging of 
adult zebrafish eye (Danio rerio). Zebrafish, a popular 
aquarium fish, has emerged as a powerful new tool to un-
derstand ocular development and a v riety of human dis-
eases like retinal detachment and blindness5–8. Th  retina 
and lens of zebrafish eye show similar morphology as those 
of other vertebrates, including humans. Various mutants 
have been identified in zebrafish that have relevance to 
human disease like retinal defects. For studies in these 
areas, measurement of several ocular parameters of the 
zebrafish, like refractive index of the crystalline lens and 
mean retinal thickness is required. Presently, most of these 
measurements are being carried out on extracted eyes. We 
have, therefore, investigated the use of OCT for non-
invasive in situ imaging of intact zebrafish eye. The use 
of OCT images to measure important ocular parameters 
like corneal thickness, effective refractive index of the 
crystalline lens and retinal thickness is discussed. 
 Figure 1 shows a schematic of the OCT set-up. The 
output of the superluminescent diode (SLD) with a centre 
wavelength of ~ 840 nm was coupled into a fibre optic-
based Michelson interferometer using a 2 ´  2 beam splitter 
designed for the wavelength used. The light beam in the  
 
 
 
 
Figure 1. Schematic of experimental set-up. 
RESEARCH COMMUNICATIONS 
 
CURRENT SCIENCE, VOL. 90, NO. 11, 10 JUNE 2006 1507 
 
 
Figure 2. a, Typical interferogram acquired at a Doppler frequency of ~ 22 kHz using the set-up (bottom) and its demodu-
lated envelope. b, Interferogram of a microscopic cover slip showing two peaks.  
 
 
 
 
Figure 3. In vivo OCT image of whole eye of zebrafish under anesthesia (a) and ~ 30min after dipped in 10% formalin 
solution. (b) Size of the image is 3 mm in depth and 2.0 mm in lateral direction. 
 
 
sample arm was collimated and focused onto the sample 
with a 10X microscopic objective. The reference mirror 
was mounted on a linear translation stage and scanned 
back and forth with uniform velocity, resulting in a Dop-
pler shift of 45 kHz. Light reflected from both the sample 
and refrence arms was detected by a photodiode (PD) and 
the resulting interferogram was amplified using a transim-
pedence amplifier (TIA) and demodulated using a lock-in 
amplifier. The interferogram envelope was digitized and 
acquired in a PC using a data acquisition card (DAQ). 
Lateral scanning was done using a stepper motor. Soft-
ware based on Labview was used to control the entire set-up. 
The free space axial and lateral resolution f the set-up 
were estimated to be ~ 11 and 17 mm respectively. The 
signal-to-noise ratio (SNR) of the set-up was measured to 
be ~ 100 dB. Typical image (500 ´  100 pixels) acquisi-
tion time was about 1 min. The OCT images have been 
false-colour coded such that white represents highest 
backscattering intensity. The maximum power incident 
on the sample was ~ 0.5 mW that is well below the ANSI 
limits for ocular damage. 
 In Figure 2 a (bottom panel), we show a typical inter-
ferogram obtained using a mirror in the sample arm of the 
interferometer. The corresponding demodulated envelope 
is shown in the top panel. From this we estimate an axial 
resolution of ~ 11 mm. Figure 2 b shows the interferogram 
of a microscopic cover slip with two peaks corresponding 
to the air–glass and glass–air interfaces. From the meas-
ured peak separation of 231 mm (optical path length dif-
ference) and assuming glass refractive index ~ 1.5, we 
e timat  a physical thickness of ~ 154 mm for the cover 
slip, which is in good agreement with the value measured 
using a calliper. 
 The lateral resolution (Dx) of the set-up is given by focal 
spot ize on the sample, Dx = (4l/p) ( f /d), where f is the 
focal length of the imaging lens and d is the diameter of 
the beam on the lens. With a 10X microscopic objective, 
lateral resolution of 17 mm is estimated. 
 We used the set-up for imaging of adult zebrafish eye. 
The fish was procured from local aquarium suppliers. For 
imaging, the fish was anaesthetized using clove oil fo-
lowing the protocols given by Grush et al.9. Briefly, clove 
oil was dissolved in ethanol (1 : 10) and then mixed in 1 l 
of water in a glass tank, such that the clove oil concentra-
tion is about ~ 75–100 ppm. The fish lost physical move-
ment within 10 min. When the fish showed no response 
to physical stimulus, it was transferred to a petri dish for 
imaging. After imaging, the fish was observed to revive
within ~ 1 h after transfer to a freshwater tank. Due to 
close index matching of cornea and water during imag-
ing, the fish eye had to be slightly projected outside the 
water while the gills remained in water. In Figure 3 a we 
show the in vivo OCT image of the whole eye of the 
anesthetized zebrafish kept in water. While the cornea, 
the anterior angle region and the retina are clearly seen, the 
lens is not clearly visible in the image due to close index 
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CURRENT SCIENCE, VOL. 90, NO. 11, 10 JUNE 2006 1508 
matching of the lens surface with the surrounding fluid. 
During the course of several experiments carried out to 
image the lens as well, it was found that the lens could be 
imaged with better contrast after dipping the fish in a 
petri dish containing 10% formalin solution for ~ 15–30 min. 
However the fish could not be revived in this case. 
 In Figure 3 b, we show the image after the fish was 
dipped in 10% formalin solution. The lens (L) as well as 
other ocular structures like cornea (C), iris (I), and retina 
(R) are clearly visible. Figure 4 shows the in vivo image 
of the cornea of zebrafish. The image is in good agree-
ment with that published in Swamynathan et al.10 using 
light microscopy. Corneal thickness in fish is an impor-
tant parameter that is affected by environmental stress 
and infection. The maximum corneal thickness seen by the 
OCT beam was measured to be ~ 34 mm. Assuming a 
corneal refractive index of 1.33, the geometrical thick-
ness of zebrafish cornea, in the region where the OCT
light is perpendicular to the cornea, turned out to be 26 mm 
(± 11 mm). In their experiments on zebrafish cornea ex-
tracted from dissected eyeballs, Swamynathan et al.10 esti-
mated its thickness to range from 16 to 20 mm. The 
difference in the two measurements may be due to the dif-
ference in the protocol and may illustrate the usefulness 
of in vivo measurements. It should be noted that since the 
resolution of our set-up is ~ 11 mm, we could not resolve the 
epithelium layer of the cornea, whose thickness has been 
reported10 to be ~ 8 mm. Use of a ultra-high resolution 
 
 
 
 
Figure 4. In vivo OCT image of cornea of zebrafish. Size of the im-
age is 0.4 mm in depth and 0.9 mm in lateral direction.  
 
 
 
 
Figure 5. In vivo OCT image of anterior angle of the cornea with the 
iris of zebrafish. Size of the image is 1.3 mm in depth and 0.7 mm in 
lateral direction. 
OCT set-up will be required for quantitative comparison 
of corneal thickness and microstructures therein. 
The anterior chamber angle is an important parameter 
in the structure of the eye. Narrowing of this angle incre ses 
the risk of glaucoma. Noninvasive measurement of ante-
rior chamber angle is therefore vital for characterization 
of structural changes in the anterior region and changes in 
th  intraocular pressure (IOP). In vivo imaging of the an-
terior chambers of different mutant variants of zebrafish 
may help in better understanding of the genetics of glau-
coma. Figure 5 shows the in vivo OCT image of the ante-
rior region of the eye. The anterior chamber angle of the 
cornea with the iris is clearly visualized. 
 Figure 6 shows the expanded OCT scan of the zebrafish 
retina with the layered structure. Due to the small retinal 
thickness and poor axial resolution compared to histology, 
ssigning the layers to specific landmarks for distinct 
characterization is difficult. However, some broad catego-
rization is possible. The first reflective interface posterior 
to the lens at the top of the image in Figure 5 represents 
the anterior border of the retina (vitreo–retinal interface) 
with the retinal nerve fibre layer (RNFL) at the vitreous 
interface. The outermost band is thought to be that of the 
reti al pigment epithelium (RPE)–choriocapillaris complex 
showing high scattering. Assuming a retinal refractive in-
d x of 1.38 and considering the band above RPE, the 
retinal thickness has been estimated as 123 ± 12 mm. This 
is quite reasonable in comparison with the mean retinal 
thickness of ~ 85 mm for wild-type zebrafish reported by 
Link et al.11 considering the fact that their measurements 
are on resected eyes. 
 In Figure 7 we show the OCT image of the whole eye 
of a zebrafish when dipped in 10% formalin solution, 
taken at a different lateral position. Along the apex of the 
lens (when the OCT beam was exactly perpendicular to 
the cornea) some scattering inside the lens was also seen, 
indicating that the crystalline lens is of a gradient index 
type. An interesting observation was that although the lens 
of the fish eye is known to be spherical in shape, the diame-
ters of the lens as measured by OCT were different along 
the axial and lateral directions. This is because, while in 
axial direction the measured diameter is the optical length 
 
 
 
 
Figure 6. In vivo OCT image of retina of zebrafish. Size of the image 
is 1 mm in depth and 0.7 mm in lateral direction. 
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CURRENT SCIENCE, VOL. 90, NO. 11, 10 JUNE 2006 1509 
 
 
Figure 7. OCT image of whole eye of zebrafish. Size of the image is 
3 mm in axial and 2.2mm in lateral direction. 
 
of the lens (geometrical path ´  effective refractive index), 
in the lateral direction it is the true geometrical diameter. 
The difference in the ratio of the measured diameters can 
be used to estimate the effective index of refraction of the 
lens medium. From the image shown in Figure 2, effective 
refractive index of ~ 1.43 was estimated for the zebrafish 
crystalline lens at the centre wavelength of the source 
(840 nm). The crystalline lens of the fish is usually as-
sumed to be spherical with a gradient refractive index 
that varies from approximately 1.35–1.38 at the surface to 
1.55–1.57 at the core of the lens12,13. 
 The refractive index of the fish lens was also measured 
using focus-tracking method described by Wang et l.14.
The sample arm light was focused using a high numerical 
aperture (NA) microscopic objective (20X) with the focal 
plane set initially at the top surface of the lens. This was 
achieved by monitoring the intensity of the A-scan peak, 
and maximizing the corresponding peak of the lens sur-
face. Once the focus was set at the top of the lens surface, 
the objective was moved a distance Dx such that the bot-
tom surface of the lens came in focus. The corresponding 
change in the refer nce mirror position (Dy) required to 
get back the A-scan peak intensity was noted. The effectiv  
refractive index was determined using the expression14 
 
 
2
2 2 4 2 2
0
1
4( ) ,
2
y
n NA NA n NA
x
é ùDæ öê ú= + + - ç ÷ê úDè øë û
 (1) 
 
where n0 is the refractive index of the surrounding me-
dium of the tissue. Before applying this technique to fish 
lens, we used it to measure the refractive index of quartz 
window. The measured value of 1.445 @840 nm was 
found to be in good agreement with the reported value14. 
From the measurements, we estimate the effective (inte-
grated) refractive index of the fish lens as ~ 1.43 @840 nm 
within 3% rms deviation. 
 The effective refractive index can also be computed as-
suming the refractive index variation along the beam di-
rection to be of parabolic form15 given by 
 
 2 20( ) 1 / ,n z n z a= -  (2) 
where n0 is the maximum refractive index at the centre of 
the lens and a is a constant that indicates the steepness of the 
gradient in the refractive index. The effective refrac i  
index of the lens (eff) as seen by the OCT beam is then 
given by  
 
 eff
( )d
,
2
r
r
n z z
n
r
+
-=
ò
 (3) 
 
where ( )d
r
r
n z z
+
-ò  is the total optical path along the apex 
of the lens as seen by the OCT beam and r is the radius of 
the spherical lens. Using OCT, r was measured to be 
0.46 mm. The index of the lens surface is reported to be 
in the range 1.35–1.38 at 632.8 nm12,13. As the OCT reflec-
tion (@840 nm) from the lens surface was feeble (possi-
bly due to close index matching of lens with surrounding 
fluid), we assume the index at the lens surface to be 1.34. 
We calculated the effective refractive index from eq. (3) 
using different values of core index n0 in the range 1.54–
1.59. Using n0 = 1.54 yields effective refractive index 
neff ~ 1.476, closer to the experimentally measured value. 
 The relative error in the estimation of effective refra-
tive index of the crystalline lens using the OCT method 
can be estimated by  
 
 eff
eff
,xz
z x
n DD
n D D
d dd
= -  (4) 
 
where Dz is the diameter of the lens along the axial direc-
tion, Dx is the diameter of the lens along the lateral direction, 
and dDz and dDx are the uncertainties in measurement of 
the diameters along the axial and lateral directions re-
spectively. Using the values dDz ~11 mm (axial resolu-
tion), and Dx ~ 27 mm (lateral resolution with 5X objec-
tive), the relative error eff eff| / |n nd  is estimated to be 
~ 2%. Taking the measured value of neff~ 1.43, the abso-
lute error in the effective refractive index estimate is 
dneff
 ~ 0.029. The discrepancy between the estimated effec-
tive refractive index (1.43) and computed value (1.476) is 
therefore not appreciable. Further, to the best of our 
knowledge there are no experimental measurements of re-
fractive index of zebrafish lens. Even though the fish 
could not be revived after these measure nts, it did not 
require separation of the lens from the fish eye unlike the 
previous studies by Garner et al.12, who used MRI for de-
termination of gradient index of fish lens after physical 
separation. The OCT method allows the fish to be intact
for further biological/biochemical studies, if necesary. 
 In summary, single-mode fibre-based OCT set-up has 
been developed and ophthalmic imaging of adult zebrafish 
eyes has been carried out. The measured corneal and retinal 
thicknesses are in reasonable agreement with the previ-
ously reported values. The effectiv  refractive index neff 
was estimated to be ~ 1.43 (± 0.03) and was independ-



Non-invasive ophthalmic imaging of adult zebrafish eye using optical coherence tomography

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Non-invasive ophthalmic imaging of adult zebrafish eye using optical coherence tomography

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