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

Abstract

1. The fundamental problem with optical imaging is that in contrast to X -rays, optical photons are strongly scattered in the tissue, which leads to blu rring of the image. Several approaches can be used to pick out the useful image-bearing light from the background multiply scattered light 2 . These exploit the depolarization or loss of coherence of scattered light or the fact that scattered light emerges from the ti ssue in all directions and also takes a longer time to emerge compared to the unsca ttered (ballistic) or pr edominantly forward-scattered (snakelike) components. The latter essentially travels in the forward direction and so arrives earlier. C oherence gating filters-out ballistic photons with the highest image info rmation and hence can provide images with high resolution. Optical coherence tomography (OCT), the approach that exploits coherence gating for optical imaging has emerged as a rapid, non-contact and noninvasive, highresolution imaging technique and is finding clinical applic ations in ophthalmology, dermatology, etc. 3,4 . The two essential 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 refe rence mirror. Light reflected from a layer of the sa mple 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 μm. Axial scanning of the reference mirror helps record interferograms from diffe rent depths of the sample. Further, scanning of the refe rence mirror at constant speed results in Doppler shift of the reference signal. Interference of the 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 fr equency, which can be isolated from the rest of the backscattered signal by a suitable bandpass filter or lock -in detection system. Twodimensional cross-sectional images and three-dimensional tomograms of the backscattered intensity distrib ution within the sample can be obtained by recording the inte rference signals from various axial and transverse positions on the sample. Two-dimensional cross-sectional images are constructed by performing measurements of the echo time delay of light at different transverse pos itions by scanning either the light beam or the sample. Here we report the implementation of single-mode fibrebased 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 u nderstand ocular development and a variety of human diseases like retinal detachment and blindness 5–8 . The 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 di sease 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 opticbased Michelson interferometer using a 2 × 2 beam splitter designed for the wavelength used. The light beam in the