Polarization properties of single layers in the posterior eyes of mice and rats investigated using high resolution polarization sensitive optical coherence tomography

Abstract

We present a high resolution polarization sensitive optical coherence tomography (PS-OCT) system for ocular imaging in rodents. The system operates at 840 nm and uses a broadband superluminescent diode providing an axial resolution of 5.1 µm in air. PS-OCT data was acquired at 83 kHz A-scan rate by two identical custom-made spectrometers for orthogonal polarization states. Pigmented (Brown Norway, Long Evans) and non-pigmented (Sprague Dawley) rats as well as pigmented mice (C57BL/6) were imaged. Melanin pigment related depolarization was analyzed in the retinal pigment epithelium (RPE) and choroid of these animals using the degree of polarization uniformity (DOPU). For all rat strains, significant differences between RPE and choroidal depolarization were observed. In contrast, DOPU characteristics of RPE and choroid were similar for C57BL/6 mice. Moreover, the depolarization within the same tissue type varied significantly between different rodent strains. Retinal nerve fiber layer thickness, phase retardation, and birefringence were mapped and quantitatively measured in Long Evans rats in vivo for the first time. In a circumpapillary annulus, retinal nerve fiber layer birefringence amounted to 0.16°/µm ± 0.02°/µm and 0.17°/µm ± 0.01°/µm for the left and right eyes, respectively.

Keywords: (110.0110) Imaging systems; (110.4500) Optical coherence tomography; (130.5440) Polarization-selective devices; (260.1440) Birefringence.

Fig. 1

3D rendering of OCT data set and sketch of the high-resolution PS-OCT system. (A) 3D rendering of a data set acquired from a Sprague Dawley rat retina (field of view 11° × 11°) together with the sketch of the rat eye to pinpoint the location. (B) Sketch of the OCT system, SLD - superluminescent diode, PC - polarization controller, SMF - single mode fiber, PMF - polarization maintaining fiber, GM - galvanometer mirrors, QWP - quarter wave plate, HWP - half wave plate, NPB - non-polarizing beamsplitter, GTP - Glan-Thomson polarizer, PB - polarizing beamsplitter, ND filter - neutral density filter.

Fig. 2

Comparison of the human, rat and mouse eyes and retinas. (A) Sketch of human, rat and mouse eye. Rodent eyes shown both in natural size and scaled to the size of the human eye. (B) Retinal OCT scan of human, rat and mouse retina (all pigmented). Human retina scanned near the fovea. The rodent eyes are much smaller than the human eye. The rodent retina is thinner than the human retina, but not proportionally to the size of the eye. Note that the layered structure of the retina is resolved in all eyes. However, higher axial resolution is required for rodent imaging in order to distinguish all the layers. The human retina was imaged using a clinical PS-OCT system described previously [25].

Fig. 3

PS-OCT images of the retina of the pigmented Brown Norway rat (averaged over 10 B-scans). (A) OCT fundus image, field of view 28° × 28°. (B) Cross section images obtained at the position indicated by the blue line on the fundus image (reflectivity, fast axis orientation and phase retardation image accordingly). (C) Cross section images at the position indicated by the red line on the fundus image (reflectivity, fast axis orientation, phase retardation image accordingly). White arrows indicate the depolarizing effect of pigment. Blue arrows indicate the birefringent effect of the sclera. The red arrow denotes higher penetration at the location of choroid vessels. The zero delays are at the top of the images. (D) Enlargement of the red rectangle in (B).

Fig. 4

Imaging of pigmentation and depolarization by PS-OCT. Images are averaged over 10 B-scans of the retina of non-pigmented (Sprague Dawley) and pigmented (Brown Norway) rats. (A) Reflectivity and DOPU images of a non-pigmented Sprague Dawley rat, taken at the optic nerve head and in the peripheral retina. (B) Reflectivity and DOPU images of a pigmented Brown Norway rat taken at corresponding locations; blue arrows highlight higher signal beneath choroid vessels. (C) Reflectivity and DOPU images of a pigmented C57BL/6 mouse. In the DOPU images, polarization-preserving tissue exhibits values around 1 (red color), whereas depolarizing tissue exhibits lower values. The depolarizing effect of pigment is visible in the proximity of the blood vessel and in RPE and choroid. (D) Enlarged part of DOPU image of non-pigmented animal and pigmented animal, respectively, together with histological image from Brown Norway rat. On reflectivity images, red arrows show extraorbital tissue, on DOPU images white arrows mark the depolarization effect of the extraorbital tissue in the non-pigmented animal and in the proximity of the central vessels in pigmented animal. (E) Comparison of OCT reflectivity image, histological image (differential interference contrast) and DOPU image of non-pigmented rat, pigmented rat and pigmented mouse. White arrow points to choroid. In the histological images, melanin pigmentation is visible as brown color (present in RPE and choroid of pigmented animals, but absent in the RPE and choroid of non-pigmented animal).

Fig. 5

DOPU analysis in RPE and choroid. (A) Reflectivity fundus projection image and exemplary B-scan with BM segmentation in a Long Evans rat. Depolarization was analyzed in slabs indicated in green (RPE) and orange (choroid). (B) Reflectivity and DOPU images in four different rodent strains. (C) The left panel shows DOPU en face projections for a Long Evans rat. In the right panel, DOPU_mean and DOPU_min are plotted for RPE and choroid in the four rodent strains (dark column represents left eye, light column represent right eye).

Fig. 6

Birefringence estimation of RNFL. (A) On top, a representative B-scan and the segmentation of the RNFL is shown. Reflectivity, retardation, thickness and birefringence maps are shown below. Pixels with a RNFL thickness below 10 pixels are displayed in grey. On the right, an enlargement of the peripapillary retardation plot depicting the evaluation area is shown. (B) Circumpapillary plots of RNFL thickness, retardation and birefringence in left and right eyes of Long Evans rats. Different colors represent three analyzed individuals. TSNIT stands for temporal – superior – nasal – inferior – temporal regions.